- Volume 4, Issue 5, 2022

Background. Human Bocavirus (HBoV), which is an ssDNA virus of the family Parvoviridae, is responsible for 21.5 % of childhood respiratory tract infections (RTIs) annually. Among the four genotypes currently known, HBoV-1 has been associated with acute RTI. Although there have been studies on HBoV in some countries, there is limited information on this virus in sub-Saharan Africa where there is the highest burden of RTI. This study aimed to characterize the circulating strains of HBoV in Ibadan, Nigeria.

Methods. Nasopharyngeal and oropharyngeal swab samples were collected from 333 children ≤5 years old presenting with RTI attending hospitals in Ibadan, whose parents assented, from 2014 to 2015. Twenty-three HBoV isolates were sequenced after a nested PCR and phylogenetic analysis was carried out using mega 6 software.

Results. A total of 27 children tested positive for the HBoV-1 genotype by PCR and 23 of the 27 isolates were successfully sequenced. The 23 HBoV-1 isolates from this study have been assigned GenBank accession numbers KY701984–KY702006. Phylogram analysis indicated that the isolates belong to the same clades. Six isolates aligned closely to the reference strains ST1 and ST2, while 17 isolates showed a high level of divergence to the reference isolates.

Conclusion. This study highlights the contribution of HBoV to RTIs in Nigeria and that HBoV-1 strains are associated with the infection.

We report the genome sequence of Streptomyces goldiniensis ATCC 21386, a strain which produces the anti-bacterial and anti-virulence polyketide, aurodox. The genome of S. goldiniensis ATCC 21386 was sequenced using a multiplatform hybrid approach, revealing a linear genome of ~10 Mbp with a G+C content of 71%. The genome sequence revealed 36 putative biosynthetic gene clusters (BGCs), including a large region of 271 Kbp that was rich in biosynthetic capability. The genome sequence is deposited in DDBJ/EMBL/GenBank with the accession number PRJNA602141.

When grown in high salt concentrations, halophilic bacteria often accumulate compatible solutes, which have major applications in biotechnology because they stabilize cells and proteins. Four Gram-negative bacterial strains, belonging to the family Halomonadaceae, were isolated from Qaberoun and Um-Alma lakes in South Libya using high-salinity medium. The strains were identified using 16S rRNA gene sequencing as belonging to Halomonas pacifica (strain ABQ1), Halomonas venusta (ABQ2), Halomonas elongata (ABU1) and Halomonas salifodinae (ABU2). H. pacifica ABQ1 is a moderate halophile (salinity range 0.05 to 2.5 M NaCl), with a broad tolerance to pH (7 to 9) and temperature (25–37 °C). Addition of the compatible solutes glycine betaine (betaine) and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) to the medium had a positive effect on growth of H. pacifica at 2 M NaCl. In rich LB medium, betaine was the major compatible solute accumulated, with ectoine only being accumulated at salinities in excess of 1 M NaCl. In minimal M9 medium, betaine was not produced, but increasing amounts of ectoine were synthesized with increasing salinity, and hydroxyectoine [(4S,5S)−5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was also synthesized when the cells were grown in very high salt. We have thus identified H. pacifica as a producer of ectoine and hydroxyectoine, with more being produced at higher salinities. As industrial demand for these compatible solutes continues to increase, this system has biotechnological potential.

We used a combination of local, comprehensive strain surveillance and bacterial whole-genome sequencing to identify potential transmission events of group A streptococcus (GAS) in Houston, TX, USA. We identified pharyngeal and skin and soft tissue sources of infection as having important roles in community GAS transmission, including invasive diseases.

The QuickGene-810 Nucleic Acid Isolation System is a semi-automated extraction platform which may be used for RNA extraction. New methods were required to support the rapid increase in respiratory virus testing during the SARS-CoV-2 pandemic. The aim of this study was to assess SARS-CoV-2 RNA extraction using the QuickGene-810 kit compared to the EZ1 Advanced Extraction Platform for use on the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well RT-PCR assay. Qualitative results from all clinical samples were concordant between the QuickGene-810 and the EZ1 extraction methods, demonstrating that the QuickGene-810 kit is suitable for use in pathogen diagnostics. However, there was an average difference of approximately two cycles between the cycle threshold (Ct) values for both SARS-CoV-2 targets, suggesting that the EZ1 kit yields a higher concentration of nucleic acid extract, possibly related to its use of carrier RNA and/or smaller elution volume, which infers the possibility of false negative results for samples with very low viral loads.

A novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suddenly emerged in China in 2019, spread globally and caused the present COVID-19 pandemic. Therefore, to mitigate SARS-CoV-2 infection effective measures are essential. Chlorous acid (HClO2) has been shown to be an effective antimicrobial agent. However, at present there is no experimental evidence showing that HClO2 can inactivate SARS-CoV-2. Therefore, in this study, we examined the potential of HClO2 to inactivate SARS-CoV-2 in presence or absence of organic matter and the results were compared with that of sodium hypochlorite (NaClO), another potent antimicrobial agent. When concentrated SARS-CoV-2 was incubated with 10 ppm HClO2 for 10 s, viral titre was decreased by 5 log of 50% tissue culture infective dose per mL (TCID50 ml−1). However, the same concentration of NaClO could not inactivate SARS-CoV-2 as effectively as HClO2 did even after incubation for 3 min. Furthermore, 10 ppm HClO2 also inactivated more than 4.0 log of TCID50 within 10 s in the presence of 5 % fetal bovine serum used as mixed organic matters. Our results obtained with HClO2 are more effective against SARS-CoV-2 as compared to NaClO that can be used for disinfectant against SARS-CoV-2 .

Introduction. Ralstonia mannitolilytica is a rare opportunistic pathogen capable of causing a serious infection in immunocompromised patients. Our objective was to describe all cases of R. mannitolilytica bloodstream infection identified within 2 years at our tertiary care centre, focusing on clinical characteristics, risk factors, antibiotic sensitivity patterns, management and outcomes.

Case Series. We compiled a descriptive case series including 14 non-duplicate R. mannitolilytica isolates obtained from bloodstream infection samples from the microbiology laboratory of a tertiary care centre from June 2019 to June 2021. All isolates were initially identified based on their morphological properties and biochemical reactions, and then underwent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) examination for confirmation of identity. Antibiotic susceptibility testing was performed using the Kirby–Bauer disc diffusion method and Vitek 2. All 14 patients presented with symptoms of fever and/or chills, and a positive blood culture for R. mannitolilytica . After 48 h of incubation, no Ralstonia growth was reported from any of the current environmental or pharmaceutical water samples. Chemotherapy (9/14), mechanical ventilation (4/14), steroid use (2/14) and diabetes mellitus (1/14) were associated risk factors in our patients. The antibiotic sensitivity panel showed maximum resistance to aminoglycosides (64.3%) and no resistance to cefoperazone/sulbactum. Patients received treatment with cefoperazone/sulbactum and meropenem or ceftazidime. Thirteen patients recovered with antibiotic therapy and one patient succumbed to his illness.

Conclusion. R. mannitolilytica can cause bloodstream infections in immunocompromised patients. It is likely to be missed or underreported due to lack of clinical awareness. MALDI-TOF MS is helpful in rapid identification. R. mannitolilytica is resistant to many routinely used antibiotics, including carbapenems.

Capnocytophaga canis is still a rare cause of infection. We present a case of an immunocompetent patient admited in the hospital with functional impotence, pain and erythema in his left leg after suffering two scratches from his cat 48 h ago. After obtaining blood and wound cultures, broad-spectrum antimicrobial therapy with intravenous amoxicillin clavulanate was initiated. After 1 day and with a clear improvement of the symptoms the patient was discharged from the hospital with cellulitis and transient bacteremia as diagnosis and completing 1 week of antimicrobial therapy orally. After 80 and 92 h of incubation, both anaerobic flasks were positive. In the Gram-stain Gram-negative rod-shaped bacteria could be observed. Despite subculturing in brucella blood agar, tripticase soy agar with 5 % of sheep blood and chocolate agar, in both anaerobic and microaerophilic conditions, the strain could not be recovered. However, these Gram-negative rods could be identified as C. canis by 16S rRNA sequencing, Capsular typing was performed to study the strain, but none of the studied capsule-types tested positive. C. canis is still a rare cause of human infection, but it must be considered in the differential diagnosis of infections related to bites, scratches and licks from dogs or cats.

Capnocytophaga canimorsus is a Gram-negative zoonotic pathogen capable of causing serious infection following dog or cat bite. Infections often manifest as sepsis, fatal septic shock, gangrene, bacteraemia, meningitis and endocarditis. Here we report a case of C. canimorsus bacteraemia complicated by tricuspid valve infective endocarditis and septic pulmonary emboli.

Cerebral venous sinus thrombosis is a rare complication of cranial melioidosis. We report a case of an adult male who presented with skull osteomyelitis, transverse sinus thrombosis and multiple brain abscesses. His blood cultures grew Burkholderia pseudomallei . The patient finally succumbed after multiple recurrences of the infection despite surgical excision of the osteomyelitic bone and the recommended antibiotic treatment. The management of cerebral venous sinus thrombosis in patients with cranial melioidosis is discussed along with a brief review of the literature.

Microbial activity is adamant for the nutrient cycling in soil. Generally, mineral phosphorus (P) fertilizer is applied to soil to improve plant growth, however, significant amounts are immobilized quickly. Mineral fertilizer can cause soil degradation, affecting the microbial community. Alternative, recycling-derived fertilizers (RDFs) need to be evaluated as suitable replacement for finite mineral P fertilizer. The impact of four RDFs (two ashes, two struvites) on the soil microbiome in comparison with a P-free control and triple superphosphate (TSP) as mineral fertilizer was investigated in a pot trial and a subsequent microcosm trial (subset of samples). For both experiments, perennial ryegrass was cultivated for 54 days. The pot trial was conducted at P fertilization rates of 20 and 60 kg P ha-1 in quadruplicates. After the pot harvest, the bulk soil was stored until the microcosm trial was conducted, using the control, TSP and the two ashes at 60 kg P ha-1 in six replicates. Struvites displayed highest P bioavailability at high P application rates in the pot trial, yielding higher biomass on average. Furthermore, P solubilization from tri-calcium phosphate was enhanced in the RDFs treatments, while the TSP treatments were negatively affected. For the microcosm trial, most probable number (MPN) analysis showed that phytate-utilizing bacterial abundance was significantly increased in one of the ashes. Non-metric multidimensional scaling (NMDS) analysis of phoD illumina sequencing data showed significant separation between all treatments of the microcosm trial. Understanding the impact of RDF application on the soil P cycle is vital to sustainable agriculture.

Zika virus (ZIKV) is an emerging flavivirus primarily transmitted through the bite of Aedesmosquitoes. It spread explosively around the tropics in the 21st century, and still presents a threat to human health. There is a need to better understand the drivers of emergence of ZIKV and likely consequences of its future evolution. RNA recombination is a process of genetic exchange which occurs in positive-sense RNA viruses and contributes to virus evolution. We have developed a recombinant-specific PCR assay for detection of ZIKV recombinants in mammalian and insect cell co-infections. Using this system, we demonstrated the ability of distinct geographic and genetic isolates to recombine and tested their viability in tissue culture. Initial ZIKV recombinants produce plaques with a significantly smaller diameter than either parental strain. Recombinant large-plaque variants were isolated through serial passage and plaque purification, and the genome analysed for adaptive mutations. A single nucleotide coding mutation in NS1 was detected in several independent large-plaque variants. The role of this mutation in virus replication was investigated through reverse genetics to assess whether it gave the recombinant a replicative advantage. Understanding the genetic changes that arise during, and following recombination is important in the context of virus evolution. It is particularly relevant for ZIKV, as co-circulation of the African and Asian strains of ZIKV in nature has already occurred in parts of Latin America and Africa. Current and future studies will focus on the phenotype of recombinant ZIKV, their replication in mammalian and insect cell lines, and in mosquitoes.

Exoribonucleases from the RNB-family of enzymes are widely distributed in nature. DIS3 is the eukaryotic homolog of the bacterial exoribonuclease II and is the only catalytic subunit of the core exosome complex. In humans, there are three members of DIS3 family that can be distinguished according to the sequence conservation of the active site:

DIS3, DIS3L (DIS3L1) and DIS3L2. Unlike its family counterparts, DIS3L2 does not interact with the exosome since it lacks the PIN domain, which is essential for the interaction with this multiprotein complex. Dis3L2 is involved in several cellular mechanisms, such as apoptosis, cellular differentiation and proliferation and its mutations have been associated with Wilms tumor formation and Perlman syndrome in children. Distinct studies on Dis3L2 enzyme unraveled a novel eukaryotic RNA decay pathway that challenged the models already established. Dis3L2 activity is stimulated by the addition of untemplated uridine residues to mRNAs, tRNAs, microRNAs, snRNAs among other classes of RNA.

The first insight on the uridylation involvement in controlling the stability of poly(A)-containing mRNAs was reported in S. pombe. However, the precise mechanism of action of this enzyme is not yet fully understood. In this work, the activity of fission yeast Dis3L2 mutant proteins was analyzed over different RNA substrates. The aim was to characterize the amino acid residues that distinguish Dis3L2 substrate specificities regarding its family homologues, namely the preference for uracil residues. The results show that some of the mutant Dis3L2 ribonucleases lose or acquire activity regarding the degradation of different RNAs. Furthermore, this will enable us to understand the mechanism of action of Dis3L2 and its function in different eukaryotic cells.

Only two patients have ever been cured of HIV-1. The latent HIV-1 reservoir is the major roadblock to this and necessitates lifelong therapy. In the ‘shock and kill’ approach to eliminate cells harbouring dormant virus the latency reversing agent (LRA) vorinostat increased HIV-1 RNA levels, but this failed to enable a reduction in reservoir size by immune- or virus-mediated cytotoxicity. To leverage LRAs’ ability to heighten HIV-1 transcription, we are developing a ‘kill’ strategy that hijacks the HIV-1 splicing process with a therapeutic trans-splicing RNA, encoding an incomplete Herpes simplex virus thymidine kinase (HSV-tk) that gains functionality by trans-splicing onto HIV-1 tat pre-mRNA. HSV-tk activates the exogenous prodrug ganciclovir for selective killing of HIV-1-infected cells. Following proof-of-principle transfection studies, therapeutic constructs were engineeredfor lentivirus-mediated delivery to HIV-infected cells in vitro. We optimized lentiviral production for high infectious titer (>106 TU/mL) and low vector plasmid carryover (<1 copy/cell). In a tissue culture model of HIV-1 infection we confirmed lentiviral delivery of therapeutic (and control) vectors. Successful trans-splicing of the HSV-tk RNA onto HIV-1 RNA in cells co-transduced with HIV-1 and therapeutic vector at an MOI as low as 4 was confirmed by sequencing. In MTT assays the most potent therapeutic vector killed approximately 80% of HIV-1-expressing cells. Next-generation vectors are being evaluated for enhanced therapeutic potential and improved HIV-1-targeting RNA expression. The lead candidate culminating from this work will be assessed for selective killing of HIV-1-infected cells both productively infected and with virus reactivated from latency.

In recent years it has become apparent that prokaryotic genomes contain large numbers of pseudogenised genes which may provide valuable insights into the recent functional history of an organism. However, pseudogenes are difficult to detectab initioand are not routinely reported by gene prediction tools.

We present StORF-R(Stop-ORF-Reporter), a tool that takes as input an annotated genome and returns putative missed genes (functional and/or pseudogenised) from the intergenic regions. We show that this methodology can recover gene-families that the state-of-the-art methods continue to misreport or completely omit.

We applied StORF-R to the intergenic regions of2,665E. coligenomes and found on average 244 previously missed pseudogenised genes (with in-frame stop codons) per genome, many of which had high scoring similarity to known Swiss-Prot proteins. Many of these pseudogenised genes form widespread gene families across E. coli strains.

To investigate if this phenomenon exists in other taxa we further applied the methodology to 44,048 bacterial genomes representing 8,244 species from Ensembl. This revealed manygene-families spanning multiple species with large (>10,000) numbers of copies of both intact and pseudogenised versions. Many of these families had only previously been reported in a single or few genomes, though we detected many hundred pseudogenised versions with StORF-R, changing our understanding of how widespread these genes truly are.

These pseudogenised genes represent a pangenomic ‘graveyard’ which may alter our understanding of the definition of core and accessory genes for many species.

Human cytomegalovirus (HCMV) is one of the most widespread, highly successful herpesviruses, establishing a life-long viral infection in humans. HCMV has been described as a paradigm of immune evasion able to manipulate many immune functions in the host. Here, we describe a novel, post-translational mechanism in which HCMV downregulates a disintegrin and metalloproteinase 17 (ADAM17), a ‘sheddase’ that cleaves and releases over 80 membrane-anchored cytokines, cell adhesion molecules and other receptors. A screen of HCMV deletion mutants identified UL148 and UL148D as the HCMV genes responsible for ADAM17 downregulation, working synergistically to alter ADAM17 levels in infected cells. We demonstrate that UL148/UL148D interfere with ADAM17 maturation, resulting in expression of only the intracellular immature precursor, and absence of mature ADAM17 on the surface of wildtype HCMV-infected cells. The consequences of ADAM17 downregulation by HCMV were analysed using proteomics and validated using biochemical and flow cytometric techniques, revealing impact on multiple cell surface and secreted host proteins. This included stabilisation of surface TNF Receptor 2, as well as Vasorin and Jagged1, which have recognised roles in Treg development. Other known ADAM17 targets were not stabilised, suggesting specific control by HCMV. Vaccinia virus, another paradigm of immune evasion, also impaired surface ADAM17 expression, suggesting that manipulation of ADAM17 may represent a novel immunoregulatory hub targeted by large DNA viruses.

The human disease cholera, marked by acute, voluminous watery diarrhea, is caused by the gram-negative, aquatic bacterium Vibrio cholerae. All seven cholera pandemics since 1817 were identified as being caused by just 2 of over 155 known V. choleraeserogroups: O1 and O139. The O1 serogroup is divided into two biotypes: classical and El Tor. Classical biotype is associated with pandemics 1 through 6, but the El Tor biotype has since displaced classical as the causative agent of the ongoing 7th cholera pandemic over the past 60 years. The El Tor genome resembles that of classical but has acquired two unique pathogenicity islands known as Vibrio Seventh Pandemic (VSP) -1 and -2. El Tor biotype has been associated with prolonged colonization, infection, and disease both in humans and in the zebrafish natural host model. The zebrafish model allows for complete observation of Vibrio cholerae infection in a system undisrupted by antibiotic use or immune suppression. El Tor strains colonize the zebrafish intestine for up to 10 days longer than classical strains. Preliminary studies demonstrate VSP-2 is required to observe this phenotype, but VSP-1 is not. By creating targeted regional knockouts of the VSP-2 island, the specific gene(s) essential for enabling prolonged colonization will be identified and applied to the understanding of how El Tor interacts with a natural host. By identifying the genes used by El Tor to colonize a natural host for prolonged periods, we gain insight into how this pathogen may persist in the environment and perpetuate disease.

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually-transmitted infection gonorrhoea, and has developed resistance to all classes of antimicrobials. In gonococci, plasmids can mediate high-level antimicrobial resistance (AMR) to tetracyclines and ß-lactams. Plasmids can spread through bacterial populations by transformation and conjugation, resulting in the rapid dissemination of traits. Characterisation of plasmids, including understanding their distribution in bacterial populations, is therefore key to understanding bacterial evolution, and in particular the spread of AMR. N. gonorrhoeae can harbour three plasmids, conjugative (pConj), ß-lactamase (pbla) and cryptic (pCryp). Using genomic and phylogenetic analyses, we show that plasmids are widespread in a large collection of gonococcal isolates from 56 countries. We found that variants of pConj (which can mediate tetracycline resistance) and pbla expressing TEM-135 ß-lactamase are associated with distinct gonococcal lineages. Furthermore, AMR plasmids are significantly more prevalent in gonococci from less wealthy countries. Over 94% of gonococci possess the cryptic plasmid (pCryp), and its absence can be correlated with the presence of a novel chromosomal Type IV secretion system. Our results reveal the extent of plasmid-mediated AMR in the gonococcus, particularly in less wealthy countries, where diagnostic and therapeutic options can be limited, and highlight the risk of their global spread.

Daptomycin is a last-resort antibiotic for the treatment of invasive diseases caused by methicillin-resistant Staphylococcus aureus. However, despite potent activity in vitro,daptomycin fails to resolve up to 30% of cases of staphylococcal bacteraemia, suggesting that the host environment reduces bacterial susceptibility to the antibiotic. Using human serum as a model of bacteraemia, we demonstrated that the host environment induced daptomycin tolerance via activation of the GraRS two-component system. Testing of various antimicrobial peptides present in serum revealed that the human cathelicidin LL-37 was able to bind GraS, induce GraRS signalling and confer daptomycin tolerance. Activation of GraRS by serum led to daptomycin tolerance via changes in the staphylococcal cell envelope. For example, GraRS-dependent increases in positive surface charge and peptidoglycan content occurred in serum, leading to reduced daptomycin binding. Additionally, incubation in serum led to a Cls2-dependent increase in the cardiolipin content of the membrane which contributed to daptomycin tolerance. Finally, inhibition of both peptidoglycan and cardiolipin synthesis together completely abolished acquisition of daptomycin tolerance in serum, demonstrating that these processes fully explain the tolerance phenotype. In summary, host LL-37 activates staphylococcal GraRS signalling, causing changes in the cell surface which confer daptomycin tolerance. This demonstrates how host defences can compromise antibiotic efficacy, and also provides a rationale for combination therapies to prevent the development of daptomycin tolerance and reduce rates of treatment failure.

Human noroviruses (HNV) are a prevalent cause of gastroenteritis that contribute to >200,000 deaths each year and cost >£40 billion worldwide per annum. There is currently no approved vaccine or therapy, and a greater understanding of the virus life-cycle could help develop new approaches towards disease control. Although HNV infection is usually self-limiting, persistent infections can establish in immunocompromised people - however the underlying mechanisms are poorly understood. Our studies use the murine norovirus (MNV) model system to investigate fundamental virus biology, and several strains of MNV can also persist in the murine host. The primary receptor for MNV, CD300lf, interacts with a network of amino acids (AAs) on the protruding domain of the virus major capsid protein (VP1). We hypothesised that genetic variations leading to changes within this network of AAs could influence the VP1-CD300lf interaction and viral persistence.

Bioinformatic analysis of the VP1-receptor interface highlighted variation in just a single AA that correlates with persistent MNV strains. To confirm this AA is important for receptor interactions we conducted in vitro evolution experiments on suspension or adherent grown cells. Passage through suspension cells resulted in the selection of hydrophobic residues at this position co-incidental with a 1.5-fold increase in viral titre. In contrast, small polar residues were maintained at this position during passage on adherent cells. Furthermore, infectivity assays with infectious clones suggest that hydrophobic residues favour infection of suspension cells over adherent cells. Work is ongoing to understand the importance of this AA on viral infectivity and persistence.

Mycobacterium tuberculosis remains the leading cause of mortality from a single infectious organism, infecting nearly one-third of the global population. The current emergence of multidrug-resistant strains represents a serious health problem nowadays. Moreover, regulation of gene expression through RNA metabolism is a key mechanism for bacterial growth, division and rapid accommodation to environmental conditions. Ribonucleases are enzymes present in all living organisms that play an important role in RNA processing and degradation. Particularly, ribonucleases belonging to the RNB-family are often essential for viability of prokaryotes and are implicated in the establishment of virulence of several pathogens. In the present study we aim to structurally and functionally characterize two putative exoribonucleases from the RNB-family of enzymes inM. tuberculosis. Overexpression and purification of the proteins was performed and further in vitro activity, binding and helicase assays using synthetic RNA substrates were accomplished. In parallel, a biophysical characterization proceeded with several crystallization trials and protein stability tests. We have demonstrated that both RNases are 3’-5’ exoribonucleases with different degradation properties and unravelled the importance of highly conserved residues for catalysis. Moreover, we were able to identify improved buffer formulations that increase protein stability, possibly enhancing their propensity to crystallize. The information regarding RNA metabolism in M. tuberculosis is limited and RNB-family enzymes have not been previously characterized in this important human pathogen. Thus, a complete knowledge of these ribonucleases is an approach to recognize their influence in M. tuberculosis metabolism and to better understand the post-transcriptional control in this pathogen.

The Aedes aegypti mosquito is a major vector of chikungunya virus (CHIKV), which has no licensed vaccine. Engineered mosquitoes expressing long RNA hairpins (lhRNA) or small RNAs against selected arboviruses have been developed to limit virus replication, but their silencing efficiency has not been compared.

We developed lhRNA and short hairpin RNA (shRNA) arrays against CHIKV non-structural protein nsP2. We used a Tet-response element (TRE) and a tTA transactivator to control expression of lhRNAs (TRE-lhRNA) and shRNAs (TRE-shRNA). Constitutive expression in Aag2 cells was assessed with a PUb-tTA driverand midgut specific expression in transgenic mosquitoes was assessed using Carboxypeptidase A (AeCPA)-tTA as driver. In vitrointerference ability was determined with a CHIKV split replication system, and a synthetic luciferase reporter with mRNA containing the targeted CHIKV sequence (CHIK-FF). In vivo interference was tested by inserting the TRE-lhRNA and TRE-shRNA constructs into a AeCPA-tTA line so both constructs expressed from the same locus, and a TRE reporter line that expressed an AmCyan reporter with an N terminal CHIKV fusion (CHIK-AmC).

In Aag2 cells, shRNAs were more effective than lhRNA in silencing both the CHIKV split replication system (99.7%, S. D. ± 0.47 and 72.8%, S. D. ± 9.50, respectively, P<0.001) and CHIK-FF (98.8%, S. D. ± 0.71 and 50.9%, S. D. ± 7.92, respectively, P<0.05). Similar results were observed in transgenic mosquitoes when comparing AmC expression in the midgut.

This study demonstrates that, in mosquitoes, effectively chosen shRNAs can induce greater interference of the desired viral target than the corresponding lhRNA.

Multidrug-resistant pathogens have become a global threat. In this context, filamentous Actinobacteria has been proven to be an exceptional source of antimicrobial metabolites. In particular, rare actinomycetes isolated from marine environments have been proposed as a potential source of yet untapped specialised metabolites. In this study, two novel species, Pseudonocardia abyssalis sp. nov. and Pseudonocardia oceani sp. nov, isolated from deep Southern Ocean sediments are described, both in terms of their phenotypic and genomic characterization. Furthermore, the genomic architecture, with a focus on Biosynthetic Gene Cluster (BGC), across eight strains belonging to the two novel species were investigated. A total of 14 Gene Cluster Families (GCF) were identified, of which five GCFs comprise BGCs from both species, and nine were specific to each species. Moreover, a correlation of GCFs to phylogeny was observed. Following genome analysis, a comparative mass-spectrometry based metabolomics analysis was carried out with one strain from each new species, as well as Pseudonocardia ammonioxydans and Pseudonocardia sediminis, also of marine origin. The metabolomics profiles agreed with the GCF distribution, where a group of ubiquitous metabolites were produced by both new Pseudonocardia spp., while groups of species-specific metabolites were also detected. This metabolic-repertoire was found to be elicitated through the addition of N-acetyl glucosamine (GlcNAc), revealing chemically-inducible bioactivity against the fungi Candida albicas and multidrug-resistant Candida auris. These results showcase the power of a combined genomic-metabolomics approach to investigate rare-actinomycetes from understudied locations and have uncovered a wealth of both biosynthetic and chemical diversity for further investigation.

Many bacterial species secrete polymers and form mat-like structures. These mats can be useful (e.g., in bioremediation), or problematic (e.g., in hospital settings). The molecular bases of mat formation have been investigated in a number of species, including various pseudomonads. One well-characterized example is the plant symbiont Pseudomonas fluorescens SBW25. Laboratory populations of SBW25 readily acquire mutations in one of three regulatory loci (wsp, aws, mws), leading to the over-production of the secondary messenger cyclic-di-GMP. In turn, this activates the production of a cellulose-like polymer, the major structural component of the SBW25 mat. Here, we dissect and compare the molecular mechanisms of mat formation in two further plant-associated pseudomonads: P. simiae PICF7 and P. fluorescens A506. We find that both PICF7 and A506 are capable of mat formation in the laboratory, by distinct molecular routes. Mat formation in PICF7 involves mutations in wsp, aws, or mws that serve to activate the production of Pel (as opposed to cellulose in SBW25). Contrastingly, A506 mat formation does not require mutation of wsp, aws, or mws (despite their retention in the genome). Instead, our results are consistent with a readily reversible, non-mutational route to polymer production and mat formation in A506. Overall, our results demonstrate the presence of multiple molecular routes to mat formation among environmental, plant-associated pseudomonads.

The presented work has since been published: Mukherjee A, Dechow-Seligmann G, Gallie J. 2022. Molecular Microbiology 117(2): 394-410.

Hepatitis E Virus (HEV) is one of the leading causes of acute viral hepatitis, with ~20 million HEV infections worldwide per annum, and mortality rates up to 25% in pregnant women. However, many aspects of the biology of the virus are poorly understood. HEV has a positive-sense single-stranded RNA genome. ORF1 encodes the non-structural polyprotein required for viral RNA replication. This polyprotein (sometimes termed pORF1) is predicted to contain seven domains based on sequence homology to related viruses and it is hypothesised that this polyprotein must undergo proteolysis to generate functional protein units. However, it is unknown if the pORF1 polyprotein undergoes full proteolysis, the potential locations of any cleavage boundaries and whether a viral or host cell protease is responsible.

We have adapted our in vitro-based proteolysis assays to investigate cleavage of HEV pORF1. In comparison to related RNA viruses, our data suggest that pORF1 has no auto-catalytic activity. Previous studies have shown that the liver-produced protease, thrombin, is essential for replication. In the presence of thrombin, we have shown that the ORF1 polyprotein undergoes specific proteolysis to produce eight distinct protein products. Combining bioinformatics with pORF1 truncations/mutagenesis, we have located the position of the pORF1 thrombin cleavage sites. Interestingly, these cleavage sites correspond to the junctions between the predicted pORF1 protein domains. Our data suggests that thrombin is an important cellular protease for controlling pORF1 proteolysis. Work is ongoing to understand the importance of each thrombin cleavage site in viral replication.

Escherichia coli is a Gram-negative bacterium that colonizes the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and some commensal E. colistrains. It can degrade mucins in the intestine, promotes biofilm maturation and in virulent strains, it is a major determinant of infection, although how it carries out these functions is not well understood. Here we examine SslE from the E. coli Waksman and H10407 strains and using electron microscopy (EM), small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy and biochemical analyses we show that SslE has a highly dynamic structure in solution. We also directly observe acidification within mature biofilms, describe a mechanism where SslE forms unique functional fibres under these conditions and determine that these SslE aggregates can bind cellulose, a major exopolysaccharide of many E. coli biofilms. Our data indicates that the spatial organization of SslE polymers and local pH are critical for biofilm maturation and SslE is a key factor that drives persistence of SslE-secreting bacteria during acidic stress.

Wagashi, also called Gassiréin the local Fulfulde language, is a traditional cheese produced from cow milk in Benin. For its preparation, milk is heated and coagulated with Calotropis procera extract. After coagulation, the heated curd obtained is drained, moulded and stained, or not, using Sorghum vulgare or Sorghum codatum panicles. It is the most commonly consumed dairy product as surrogate for meat or fish. In order to get better insights into the bacterial diversity of Wagashi, Illumina MiSeq amplicon sequencing targeting the V1-V2 region of the bacterial 16S rRNA gene was performed on two different Wagashi types (stained and not stained) from Benin. The results showed that the Lactobacillaceae (60%) and Streptococcaceae (38%), were the most abundant bacteria in the unstained cheese, whereas Streptococcaceae (89%) and Bacillaceae (10%) were the most prevalent in stained cheese. Moreover, at the species level, the microbial community structures of stained and unstained cheeses were significantly different. The main differentiating species were Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus fermentum in unstained cheese, compared to Streptococcus infantarius, Aeromonas sp., Bacillus cereus, Bacillus subtilis and S. thermophilus in stained cheese. This work provides valuable insights into microbiology of cheese Wagashi and shows that the staining of this dairy product affects its bacterial composition. In particular, the presence of potential useful Lactobacilli and Streptococci in unstained cheese is to be further investigated for selecting functional bacteria for potential biotechnology applications. On the contrary, the presence of Bacilli in stained cheese needs additional study to detect potential foodborne strains or pathotypes.

Cells require thousands of unique proteins to be in the right place, at the right time, in the right amounts and with the right modifications. They do this through several processes collectively known as protein homeostasis. TORC1 is a principal regulator of protein homeostasis, coordinating protein synthesis and degradation. The proteasome, composed of a core particle and one or two regulatory particles, degrades unwanted protein. Diverse stresses cause a protein homeostasis imbalance: inhibiting TORC1 and the misfolded/damaged protein load. Following TORC1 inhibition, proteasome regulatory particle assembly chaperone (RPAC) translation is increased and thus cells assemble more proteasomes to degrade the damaged and misfolded proteins, thereby restoring protein homeostasis.

Using yeast, we identify an endocytic protein, Ede1, that interacts with and is critical for translation of RPAC mRNA following TORC1 inhibition. We find two further endocytic proteins important for RPAC translation regulation. Mutants of these proteins cause altered Arp2/3 activity, and hence altered formation of actin patches/endocytic sites. We show that RPAC mRNA is transported on actin cables and interacts with actin patches. TORC1 inhibition depolarises the actin cytoskeleton, causing RPAC mRNA accumulation on actin patches concurrent with translation. We demonstrate Ede1 is essential for RPAC mRNA localisation regulation following rapamycin treatment.

This work shows that, upon actin depolarisation, RPAC mRNA is recruited to actin patches, likely by Ede1, and translation occurs. Actin regulation is therefore a key element of proteasome (and therefore protein) homeostasis.

We present results demonstrating species-specificity and sub-species resolution by novel, automatically-designed metabarcoding primers for environmental DNA analysis.

Conventional metabarcoding remains a cornerstone of rapid, high-throughput environmental DNA (eDNA) community analysis and biodiversity assessment. Standard barcodes such as 16S (prokaryotes) and ITS1 (fungi/oomycetes) have been instrumental in identifying the complex composition of communities using total eDNA. However, standard barcodes have limitations in terms of resolution and quantitation and, though genus-level identification can be reliable, species-level identification is often not possible.

To overcome the limitations of resolution, we implemented extensions to the diagnostic primer design tool pdp (https://github.com/widdowquinn/find_differential_primers [https://github.com/widdowquinn/find_differential_primers]) that enable automated design of metabarcoding markers and corresponding primers that are (i) specific to a prescribed taxon at species level and (ii) capable of discriminating between members of the same species. This allows for rapid, high-throughput measurement of diversity below species level for a target organism.

We aimed to survey geographical distribution and pathogen transfer of the widespread plant pathogenic bacterium Pectobacterium atrosepticum (Pba). This organism has considerable sub-species taxonomic structure identifiable using MLST and with whole-genome methods, but which is not accessible using standard barcodes. We designed metabarcoding primers (202bp) specific to Pba using `pdp`, and established that these have resolution comparable to eight-gene MLST, revealing sub species-level diversity within single fields, and on the same individual plant host.

The Mycobacterium tuberculosiscomplex (MTBC) is a group of bacteria that show more than 99% genetic identity, yet they diverge in their host preference and the severity of associated disease. Mycobacterium bovis causes the disease in cattle and poses an economic challenge, with the cost of Irelands TB Eradication Scheme predicted to exceed €90 million in 2020.

The aim was to focus on the RD900 and pknH gene network in MTBC members. ThepknH gene encodes a transmembrane serine threonine protein kinase, that plays a role in the regulation of signalling pathways in the mycobacterial cell and has been linked to virulence. The RD900 or “region of difference 900” contains the tbD2 and pknH2 genes. The part of the genome that contains this region varies substantially across MTBC members.

The results of RNA-seq analysis showed that there are statistically significant levels of differential gene expression in wildtype and knock-in M. bovis strains.

The RD900 analysis across the MTBC showed that there is variation in this important genomic region. Analysis carried out by Mata et al. 2020, showed that this RD900 region had been independently lost in different MTBC lineages and strains. M. bovis has retained the RD900 region and this is believed to be linked to its increased virulence.

The majority of the M . africanum strains were found to possess the standard pknH, tbD2, pknH2, embR gene arrangement as is found in M. africanum GM041182, the strain that is typically used as a reference.

Based on work of Mata et al., 2020.

The amino sugar N-acetylglucosamine (GlcNAc), widely present in multiple cell-surfaces is efficiently utilized by human pathogen Candida albicans at the sites of infection as a survival strategy in different host niches. GlcNAc import inside the cell is mediated by GlcNAc transporter, Ngt1. In this present study, to investigate the Ngt1 dynamics, we have checked the sensitivity of Ngt1 for GlcNAc, expression kinetics and dynamics of Ngt1, stability of Ngt1 in the presence of unrelated carbon source like, glucose and endocytic trafficking of Ngt1. For this study we have used epi-fluorescence microscopy, Western blot and Northern blot analysis. We have observed that Ngt1 expression is prolific and highly sensitive to even minute amount (<2 μM) of GlcNAc. Ngt1 maintains its turnover in the plasma membrane by sugar stimulated endocytosis via UBI4 (polyubiquitin encoding locus) mediated ubiquitylation. Co-localization studies with different sub-cellular markers have revealed that Ngt1 follows a trafficking route via early endosome-late endosome-multi vesicular body (MVB)-Trans Golgi network for degradation. In conclusion, the study will provide a better understanding of endosomal trafficking for other membrane proteins in particular sugar transporters and also will open new therapeutic aspects in Candida albicans.

RND efflux pumps are important mediators of antibiotic resistance. RND efflux pumps including AcrB, are organized as tripartite systems, consisting of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. We have previously identified the residues required for binding of the major PAP AcrA to the major RND pump AcrB and shown that there is promiscuity between the PAPs such that they can function with non-cognate pumps. AcrE is a PAP homologous to AcrA and AcrD and AcrF are RND pumps homologous to AcrB. This study aimed to determine whether the PAP AcrE can function with AcrD, which does not have its own PAP, to mediate efflux and whether the previously identified RND binding residues in AcrA and AcrE were also required for AcrD binding. The acrD and acrE genes were cloned into compatible vectors and co-transformed into a strain lacking acrAB, acrD and acrEF. When expressed together, acrDand acrE significantly decreased susceptibility of the efflux mutant strain to AcrD substrates including aztreonam, carbenicillin, cloxacillin, fusidic acid, nafcillin, novobiocin, oxacillin and ticarcillin, indicating that AcrE can also form a functional complex with AcrD. These experiments also highlighted that the substrate profile of AcrD in S. entericaand E. coli are different. Point mutations in the previously defined RND binding residues of AcrA and AcrE impaired AcrD-mediated efflux of substrate drugs which validates the interchangeability of AcrA and AcrE and highlights these residues as ideal drug targets for efflux inhibition to combat antimicrobial resistance.

Streptococcus pneumoniae is a major cause of global morbidity and mortality. It behaves as a commensal in the host nasopharynx, but can become pathogenic, invading the lungs, blood, and meninges. As such, identification of pneumococcal virulence and colonisation factors remains a major research objective. We previously described an experimental evolution approach for the identification of pneumococcal genes that make niche-specific contributions to fitness and virulence. Sequential passage of pneumococci through mouse models of nasopharyngeal-carriage and pneumonia was performed, generating bacterial lineages adapted to the nasopharynx and lungs, respectively.

Using RNA-Seq differential gene expression analysis, this study compared the transcriptomic profile of a nasopharynx-evolved pneumococcal lineage that showed evidence of enhanced nasopharyngeal colonisation potential, with a lab-adapted ancestor strain. Here, we describe how the genomic adaptations acquired by this lineage, and which we have demonstrated facilitate survival in the nasopharynx, can influence bacterial gene expression.

One key finding was the identification of five adjacent upregulated genes, representing a putative pneumococcal operon. These poorly characterised genes are predicted to encode a carbohydrate-scavenging pathway. Expression of the operon within the nasopharynx may facilitate sugar acquisition from host glycoproteins. Of note, the nasopharynx evolved pneumococcal lineage carries a single nucleotide insertion mutation immediately adjacent to the -10 element of the operon predicted promotor sequence. This may contribute to increased operon gene expression in the nasopharynx-adapted pneumococcus, thereby enhancing colonisation potential. Confirmatory mechanistic investigations are underway, which will aid the identification of pneumococcal virulence factors involved in the commensal to pathogen switch.

There is an urgent need for new antimicrobials due to constantly advancing antimicrobial resistance. Here, we worked with environmental samples from diverse habitats including different savannah and forest soils, volcanic caves, and termite mounds and assessed their microbial communities for the potential of biosynthesis of secondary metabolites. We analysed and compared microbial composition by applying the QIIME2 pipeline to 16S rRNA gene data. We focused on the abundance of Actinobacteria and Streptomyces as they are important producers of antimicrobials. Out of the samples analysed, the highest abundance of Actinobacteria was found in termite mound and volcanic cave samples. Moreover, the termite mound samples also had the highest abundance of Streptomyces. When comparing microbial composition, soil samples and termite mound samples each formed their own clusters, but volcanic cave samples appeared more dispersed. We assessed the antimicrobial potential of a subset of samples by analysing metagenomic data to predict biosynthetic gene clusters (BGCs) using antiSMASH5.2.0, which resulted in over 800 hits per sample. This number was narrowed down by evaluating identified BGCs based on antimicrobial potential, completeness, size, presence/absence of regulatory and transport-related genes, and dissimilarity with known BGCs. This resulted in an average of 20 BGCs per sample. These BGCs will be subjected to further sequence-based analyses before attempting heterologous expression. Following successful expression, antimicrobial potential will be assessed by screening for growth inhibition of multidrug resistant E.coli strains and the ESKAPE pathogens.

The excessive use of antibiotics in agriculture is routinely described as a major contributor to bacterial antimicrobial resistance. Globally, antibiotics are also widely used as growth supplements in livestock. This has led to concerns regarding use of human-use antibiotics in food and food-producing animals. In more recent times organic acids such as propionic acid (PA) and formic acid (FA) have been used as alternative antimicrobials or preservatives in place of antibiotics.

PA is a short chain fatty acid naturally abundant in the human and animal intestine as a breakdown product of non-digestible carbohydrates. In the human intestine it plays important roles in regulating the immune response in the human body. Recently, a study has shown that exposure of a Crohn’s Disease associated bacterial pathotype, adherent-invasive Escherichia coli(AIEC),to PA significantly altered its phenotype resulting in increased adhesion and invasion of epithelial cells and increased persistence through biofilm-formation.

AIEC are both evolutionarily and phylogenetically related to avian pathogenicEscherichia coli (APEC). PA and FA use is widespread in chickens a known source of zoonotic disease. Our results indicate that virulence of some APEC strains is increased by exposure to alternative antimicrobials such as PA and FA. This included increased adhesion of APEC strain E. coli 601 to human intestinal epithelial cells after exposure to FA which could be a potential risk of zoonotic disease. Further investigation of APEC strains is currently underway in a fermentation model of the poultry gut. This approach will improve our understanding of how commonly used.

As the seagrass leaves remain underwater, the primary source of leaf microbes is considered to be seawater heterotrophic bacterioplankton, which possess the ability to degrade biopolymers and are known to attach to surface and form biofilms. In this study, 16S rRNA gene amplicon sequencing was used to assess bacterial diversity and dynamics of the particle associated (PA) and free-living (FL) fraction of the seagrass-covering seawater (inside) and bulk seawater (outside) among different seagrass bed around Japan. Samples were collected from the three Zostera marinabeds (Ikuno-shima Is., Hiroshima; Nanao Bay, Ishikawa; Mutsu Bay, Aomori Prefecture) around Japan during summer (June-August 2015; July 2016). Prokaryotic DNA was extracted from samples using a FastDNA spin kit according to the manufacturer’s protocol. After extracting DNA, 16S ribosomal RNA (16S rRNA) genes were sequenced by Illumina Miseq platform. The Results showed that PA bacterial communities had a higher (p <0.001) diversity than FL ones. Compared to the outside of the seagrass bed, the inside had lower diversity both in PA and FL fraction. Taxonomic analysis revealed a different community composition between lifestyle (PA vs FL) and sampling point (inside vs outside). Differential abundance analysis showed that PA were significantly enriched in a diversity of Cyanobacteria (Synechococcaceae), Saprospiraceae and Hyphomonadaceae. Conversely, FL were more abundant in Gammaproteobacteria (including Halomonadaceae, Alteromonadaceae), Microbacteriaceae, Campylobacteraceae, Pelagibacteraceae,Acidimicrobiia(OCS155). The present data provide a comprehensive description of the PA and FL microbial community in the seagrass bed and can be useful for better understanding the seagrass microbe interactions.

The importance of genomics in the COVID-19 age cannot be overstated and genomics has a key place in the advanced undergraduate microbiology curriculum. The COVID-19 pandemic, and its attendant lockdowns, have necessitated a change in the delivery of our microbial genomics module. The key challenges for delivering a remote computer-based genomics module, are ensuring student engagement and learning; and supporting the technical aspects of remote computer work.

In the absence of in-person classes, we have adapted our material covering the principles and theory of microbial genomics, to asynchronous videos and synchronous online workshops. Practical training for all undergraduate microbiologists in the UK has been severely reduced, which has forced us to focus skills training towards computational aspects of genome assembly, analysis, and interpretation. The set up and implementation of a robust and scalable Linux-based genome analysis pipeline for students presents many challenges: from organising remote access to computer clusters; software support; and managing hardware.

Assessment for the module is based on the demonstrating learning and skills development through the analysis of SARS-CoV2 genomes to identify spike protein variation and mapping this to published structures. Terminal assessment is through the analysis of newly sequenced bacterial genomes and the preparation of a genome report suitable for publication.

We will outline the implementation and management of our Microbial Genomics module as a model for computer-based skills training for undergraduate microbiologists. Furthermore, we will discuss the impact of the COVID-19 pandemic on the module and the opportunities it has presented.

Pseudomonas aeruginosais an important opportunistic pathogen, causing nosocomial infections. The Liverpool Epidemic Strain (LES), a major cause of mortality and morbidity in cystic fibrosis patients, harbours five prophages associated with increased fitness and survival in models of infection. However, ~76.5% of the LES prophage genes are hypothetical proteins. Also, little is known about the LES prophage interactions with the lysogen and other prophages. In this study, we used the VIral Genome Annotation (VIGA) pipeline to re-annotate the LES prophage genomes and improve the prediction of gene function. The RefSeq viral proteins database was used for homology-based gene prediction and the Prokaryotic Virus Orthologous Genes (PVOGs) and Reference Viral DataBase (RVDB) were used for HMM-based protein function prediction. InterProScan 5.47-5.82 and Infernal 1.1.3 (with Rfam 14.3) were applied to enrich the gene function prediction for all genes and ncRNA elements, respectively. The number of putative coding sequences had increased by 1.17-1.43 times. Multiple genes related to DNA recombination and host cell lysis were identified in this reannotation. Also, we have identified new ncRNA elements in these prophages, such astRNAs in prophages 2 and 5 and a putative regulatory ncRNA, a Hammerhead-II ribozyme, in prophage 4. All this new information will be combined with data from future RNAseq experiments to map the expression profiles of each LES prophage under inducing and non-inducing conditions to characterise interactions between the prophages and their lysogen host.

Vaccines remain the primary means of disease prevention throughimmunisationschemes in the poultry sector. Novel approaches in vaccine development, such as reverse genetic systems and genome editing technologies (i.e. CRISPR/Cas9), are currently being utilized to overcome challenges in establishing an immunogenic platform that is safe and capable of inducing long-term immunity. The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats associated protein nuclease 9) technology offers an effective, fast and simple novel approach to edit genomes for the development of viral vectors against poultry diseases such as Newcastle disease (ND), avian influenza (AI) and infectious bronchitis (IB). In this study, we demonstrate the application of CRISPR/Cas9-based genome editing to generate recombinant viral vectors that express the F gene of NDV. Validation of gene integration, protein expression, and insert stability was carried out by PCR, immunostaining and Western blot analysis, respectively. This approach offers an efficient platform for the generation of multivalent recombinant vaccines that can provide simultaneous protection against major poultry diseases.

Acyltransferase-3 (AT3) domain-containing proteins are involved in acylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, antimicrobial resistance, and antibiotic biosynthesis. Despite this, the mechanism of action is largely unknown. AT3 proteins fromSalmonellaspp. are responsible for acetylation of lipopolysaccharides which can generate a specific immune response upon infection. Here we analysed two AT3 proteins fromSalmonellaspp., some differences exist but both contain an integral membrane AT3 domain fused to a periplasmic SGNH domain. Identification of essential residues from each domain suggests both domains are required for acylation.

The crystal structure of the SGNH domain and periplasmic linking region was determined. Novel structural features are seen in comparison to other SGNH domains. In particular, the periplasmic linking region is structured and forms an extension of the SGNH domain (SGNH-extension). Removal of the SGNH-extension suggests that this region is important for stability of the SGNH domain. The structure of the SGNH-extension suggests the SGNH domain is in close proximity to the acyltransferase domain and the domains may interact. In silicoco-evolution analysis, used to make predictions about the structure of both domains, suggests likely inter-domain interactions. This analysis also predicted which transmembrane helices in the acyltransferase domain interact giving an insight into the overall structure.

Combining these data we propose a refined model of AT3-SGNH proteins which enhances our understanding of the mechanism and function of AT3 proteins required for modification of cell-surface carbohydrates.

genomeRxiv is a newly-funded US-UK collaboration to provide a public, web-accessible database of public genome sequences, accurately catalogued and classified by whole-genome similarity independent of their taxonomic affiliation. Our goal is to supply the basic and applied research community with rapid, precise and accurate identification of unknown isolates based on genome sequence alone, and with molecular tools for environmental analysis.

The DNA sequencing revolution enabled the use of cultured and uncultured microorganism genomes for fast and precise identification. However, precise identification is impossible without

1. reference databases that precisely circumscribe classes of microorganisms, and label these with their uniquely-shared characteristics

2. fast algorithms that can handle the volumes of genome data

Our approach integrates the highly-resolved classification framework of Life Identification Numbers (LINs) with the speed and computational efficiency of sourmash and k-mer hashing algorithms, and the precision and filtering of average nucleotide identity (ANI). We aim to construct a single genome-based indexing scheme that extends from phylum to strain, enabling the unique and consistent placement of any sequenced prokaryote genome.

genomeRxiv includes protocols for confidentiality, allowing groups to identify and announce the identities of newly-sequenced organisms without sharing genome data directly. This protects communities working with commercially- and ethically-sensitive organisms (e.g. production engineering strains, potential bioweapons, and to enable benefit sharing with indigenous communities).

genomeRxiv will also provide online capability to design molecular diagnostic tools for metabarcoding and qPCR, to enable tracking of specific groupings of bacteria directly in the environment.

Staphylococcal pathogenicity islands (SaPIs) are mobile genetic elements encoding superantigens and other toxinsand are induced for excision, replication, packaging and intercell transfer by phage-encoded anti-repressors that counter the SaPI master repressor. Though SaPI induction has heretofore been assumed to be the exclusive province of helper phages, we report here the remarkable discovery that one of the SaPIs, SaPI3, can instead be induced only by a second, co-resident SaPI, which must first be induced by a phage. This induction cascade thus represents intricate regulatory triad; SaPI3, the beneficiary of this intracellular largess, is the prototype of a hitherto uniquely immobile SaPI lineage. We report that members of this lineage are controlled by a novel regulatory module and are induced by a highly conserved but previously uncharacterised SaPI protein. SaPI3 and its cousins are thus SaPI satellites, just as most other SaPIs are phage satellites.

Amoeba-related diseases have been related with the presence of certain amoebas in domestic water, including drinking water. Biofilms in drinking water distribution systems are able to support amoeba growth by providing a food source and protecting them against disinfectants. Additionally, amoeba growth can be favoured by warm temperatures and climate change appears to contribute to its geographic spread.

The presence of amoeba and its association with potential pathogenic bacteria was studied in a real-scale chlorinated DWDS. The test facility comprised three independent pipe loops fed with water from the local water supply and for this study a varied flow hydraulic profile was applied based on daily patterns observed in real UK distribution networks. The daily regime was repeated for a biofilm growth phase of 30 days. Amoeba viability was tested by a culture-based method, non-nutrient agar (NNA)-E. coli plates, and then confirm by qPCR using specific primers to detect species of amoeba including Naegleria and Acanthamoeba. Amplicon sequencing of the 16s rRNA gene was used to characterise the biofilm and planktonic bacterial communities.

Several amoeba species belonging to the genera Acanthamoeba, Vermamoeba and Naegleria were identified in 30-day old biofilm samples. While the bacterial communities in biofilms were dominated by Variovorax, Pseudomonas and Aquabacterium. This study yielded new insights on the dynamics of amoeba and bacterial communities in DWDS. However, more research is required to accurately establish the impact of these inter-kingdom associations on human health.