Biology subjects

Hill, J. E.

Publications and source records attributed to Hill, J. E..

18 recordsLinked to original sources

bioRxiv2025

Genomic differentiation of Mannheimia haemolytica genotypes

Mannheimia haemolytica is an opportunistic bacterial pathogen associated with the economically costly bovine respiratory disease. Two genotypes have been described, of which genotype 2 is more strongly associated with disease. Several previous studies have investigated the genomic differences between the genotypes and/or the major serotypes (1, 2 and 6) of M. haemolytica, however we still lack a clear basis for the greater disease association of genotype 2 (serotypes 1 and 6) and demonstrations of phenotypic differences are scarce. This work builds upon previous investigations to identify genes that differentiate the two genotypes with a particular focus on genes that may play a role in virulence and fitness in the respiratory tract microbiome. We identified 422 genotype differentiating genes in a collection of 206 unique M. haemolytica genomes (61 genotype 1, 145 genotype 2). Genotype differentiating genes included genotype-associated variants of a TonB-dependent siderophore receptor homolog, transferrin binding protein B, leukotoxin A, and IgA1 proteases. We also identified a genotype 1 associated lytic transglycosylase, and a genotype 2 specific highly immunogenic outer membrane lipoprotein. Genotype 2 genomes were significantly larger in size and contained more predicted protein coding genes than genotype 1 genomes. These results expand our knowledge of what differentiates the genotypes 1 and 2 of M. haemolytica and provides information that can be used as the basis for laboratory investigations of corresponding phenotypic differences.

microbiology↗

bioRxiv2025

Transformation of Gardnerella vaginalis with a Bifidobacterium-Escherichia coli shuttle vector plasmid

Gardnerella spp. significantly influence female reproductive health, and are indicators of bacterial vaginosis, a common gynecological disorder. Lack of genetic tools for Gardnerella spp. is a hindrance in fully understanding their role in the vaginal microbiome and no naturally occurring plasmids have yet been identified in these organisms. The aim of this study was to transform Gardnerella vaginalis and characterize transformants carrying Bifidobacterium-E. coli shuttle vector pKO403-lacZ'-Sp. G. vaginalis ATCC 49145 was selected for protocol development based on its high growth rate, lack of restriction activity and susceptibility to spectinomycin. Low efficiency ([~]102 cfu/{micro}g of plasmid DNA) but reproducible transformation was achieved. The expression of the spectinomycin resistance gene and the {beta}-galactosidase gene of pKO403-lacZ'-Sp in G. vaginalis ATCC 49145 resulted in an increase in spectinomycin tolerance from 2 {micro}g/ml (MIC) to >512 {micro}g/ml, and an appreciable increase in {beta}-galactosidase activity compared to the wild type. Plasmid copy number was determined to be [~]3 per genome copy. Plasmid was lost rapidly in the absence of spectinomycin selection, with only [~]13% of colony forming units retaining the resistant phenotype after 24 h of growth without selection. These results demonstrate that G. vaginalis can be transformed by electroporation and that pKO403-lacZ'-Sp can be maintained and its genes expressed in this host, offering a starting point for the development of genetic tools for mechanistic studies of this important member of the vaginal microbiome. ImportanceThe healthy human vaginal microbiome is mainly dominated by Lactobacillus spp. An imbalance or shift in this population can lead to a gynecological disorder known as bacterial vaginosis (BV). In BV, there is a reduction in Lactobacillus spp. and overgrowth of mixed anaerobes and facultative bacteria including Gardnerella spp. The reason for this increase in the Gardnerella population and associated changes in the vaginal microbiota composition is yet not understood, and a lack of genetic tools is one of the major barriers to performing mechanistic research to study the biology of these clinically significant organisms. A first step in developing genetic tools is introducing foreign DNA. In this study, we have developed a protocol for transformation and identified a plasmid that can be maintained in G. vaginalis.

microbiology↗

bioRxiv2024

Targeted syndromic next-generation sequencing panel for simultaneous detection of pathogens associated with bovine reproductive failure

Bovine reproductive failure, which includes infertility, abortion, and stillbirth in cattle, leads to significant economic losses for beef and milk producers. Diagnosing the infectious causes of bovine reproductive failure is challenging as there are multiple pathogens associated with it. The traditional stepwise approach to diagnostic testing is time-consuming and can cause significant delays. In this study, we have developed a syndromic next-generation sequencing panel (BovReproSeq), for the simultaneous detection of 17 pathogens (bacteria, virus and protozoa) associated with bovine reproductive failure. This targeted approach involves amplifying multiple pathogen-specific targets using ultra-multiplex PCR, followed by sequencing with the Oxford Nanopore platform and subsequent analysis of the data using a custom bioinformatic pipeline to determine the presence or absence of pathogens. We tested 116 clinical samples and found that BovReproSeq results matched with current diagnostic methods for 93% of the samples, and most of the disagreements occurring in samples with very low pathogen loads (Ct > 35). At the optimal read-count threshold of 10 reads (minimum number of reads to classify the sample as positive), the clinical sensitivity of the assay was approximately 82%, while clinical specificity was 100%. The overall accuracy of the assay was 98.8%. Matthews Correlation Coefficient (correlation coefficient of binary classification) was approximately 0.90 and F1 score (harmonic mean of Precision and Recall) was 0.90, indicating excellent overall performance. Our study presents a significant advancement in detecting the infectious agents associated with bovine reproductive failure and the BovReproSeq panels ability to detect 17 pathogens makes it a promising tool for veterinary diagnostics. ImportanceBovine reproductive failure causes substantial economic losses to beef and milk producers, and infectious disease contributes significantly to this syndrome. Etiologic diagnosis is complicated since multiple pathogens can be involved and infections with some pathogens are asymptomatic or cause similar clinical signs. A stepwise approach to diagnostic testing is time-consuming and increases the risk of missing the correct diagnosis. BovReproSeq is a next-generation sequencing based diagnostic panel that allows detection of 17 reproductive failure pathogens simultaneously.

microbiology↗

bioRxiv2024

Investigation of expression of genes encoding glycogen degrading enzymes in Gardnerella swidsinskii and identification of reference genes for quantitative real-time PCR

Gardnerella spp. express and export enzymes for the breakdown of glycogen into glucose, maltose, and malto-oligosaccharides for consumption by the vaginal microbiota but how the expression of these "public goods" is affected by substrate and product levels in the environment is not known. Accurate measurement of relative gene expression using real-time quantitative PCR relies on the identification of appropriate reference genes whose expression levels remain constant under the conditions of the study. Currently, no reference genes have been identified for gene expression analysis of Gardnerella spp. The objectives of this study were to identify reference genes and apply them in determining the relative gene expression levels of genes encoding -amylase and -amylase-pullulanase in media supplemented with substrate (glycogen) or a preferred product (maltotriose). Ten candidate reference genes were evaluated and analysis of Cq values from qPCR using multiple algorithms identified uppS (encoding polyprenyl diphosphate synthase) as the top comprehensively ranked reference gene followed by gatA (encoding Asp-tRNA/Glu-tRNA amidotransferase subunit gatA). Interpretation of the Cq values for -amylase and -amylase-pullulanase was performed by applying these two reference genes in the calculation of relative gene expression levels. -amylase-pullulanase gene expression was upregulated in media supplemented with 1% glycogen in comparison to media supplemented with 1% maltotriose suggesting a regulatory mechanism in G. swidsinskii that responds to nutrient availability. No significant difference in gene expression of -amylase was observed suggesting expression is not influenced by substrate availability. The RNA purification protocol and reference genes validated in this study will be useful in future studies of gene expression in Gardnerella. ImportanceKnowledge of the factors affecting growth of vaginal microbiota is critical to understanding how vaginal dysbiosis is initiated and maintained. Overgrowth of Gardnerella species including G. swidsinskii is a hallmark of bacterial vaginosis. These organisms break down vaginal glycogen and the products become available for uptake by Gardnerella and other microbiota. Measuring how expression of genes encoding glycogen degrading enzymes relates to relative abundance of substrate and products in the environment requires development of protocols for RNA purification and identification of reference genes for RT-qPCR.

microbiology↗

bioRxiv2023

Rapid and accurate taxonomic classification of cpn60 amplicon sequence variants

The "universal target" region of the gene encoding the 60 kDa chaperonin protein (cpn60, also known as groEL or hsp60) is a proven sequence barcode for bacteria and a useful target for marker gene amplicon-based studies of complex microbial communities. To date, identification of cpn60 sequence variants from microbiome studies has been accomplished by alignment of queries to a reference database. Naive Bayesian classifiers including the RDP classifier offer an alternative identification method that provides variable rank classification and shorter analysis times. We curated a set of cpn60 barcode sequences to train the RDP classifier and tested its performance on data from previous human microbiome studies. Results showed that sequences accounting for 79%, 86% and 92% of the observations (read counts) in saliva, vagina and infant stool microbiome data sets were classified to the species rank. We also established a threshold confidence value of 0.6 at phylum rank for filtering non-target amplicon sequences from study data and demonstrated that trimming the training sequences to match the lengths of the queries is not needed for accurate cpn60 sequence classification. Successful implementation of a naive Bayesian classifier for cpn60 sequences will facilitate future microbiome studies and open opportunities to integrate cpn60 amplicon sequence identification into existing analysis pipelines.

microbiology↗

bioRxiv2022

Transport and utilization of glycogen breakdown products by Gardnerella spp. from the human vaginal microbiome

Multiple Gardnerella species frequently co-occur in vaginal microbiomes, and several factors including competition for nutrients such as glycogen could determine their population structure. Although Gardnerella spp. can hydrolyze glycogen to produce glucose, maltose, maltotriose and maltotetraose, how these sugars are transported and utilized for growth is unknown. We determined the distribution of genes encoding transporter proteins associated with the uptake of glucose, maltose, and malto-oligosaccharides and maltodextrins among Gardnerella species. A total of five different ABC transporters were identified in Gardnerella spp. of which MusEFGK2I and MalXFGK were conserved across all 15 Gardnerella isolates. RafEFGK and TMSP (trehalose, maltose, sucrose and palatinose) operons were specific to G. vaginalis while the MalEFG transporter was identified in G. leopoldii only. Although no glucose specific sugar-symporters were identified, putative glucose/galactose porters and components of a phosphotransferase system were identified. In laboratory experiments, all Gardnerella isolates grew more in the presence of glucose, maltose, maltotriose and maltotetraose compared to un-supplemented media. In addition, most isolates (10/15) showed significantly more growth on maltotetraose compared to glucose (Kruskal Wallis, P < 0.05) suggesting their preference for longer chain malto-oligosaccharides. Our findings show that although putative MusEFGK2I and MalXFGK transporters are found in all Gardnerella spp., some species-specific transporters are also present. Observed distribution of genes encoding transporter systems was consistent with laboratory observations that Gardnerella spp. grow better on longer chain malto-oligosaccharides. ImportanceIncreased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community. Gardnerella produces enzymes to digest glycogen, an important nutrient source for vaginal bacteria, but little is known about the mechanisms in Gardnerella for uptake of the products of this digestion, or whether Gardnerella use some or all of the products. Our results indicate that Gardnerella may have evolved to preferentially use a subset of the glycogen breakdown products, which would help them reduce direct competition with some other bacteria in the vagina.

microbiology↗

bioRxiv2022

Glycogen degrading activities of catalytic domains of α-amylase and α-amylase-pullulanase enzymes conserved in Gardnerella spp. from the vaginal microbiome

Gardnerella spp. are associated with bacterial vaginosis, in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake; a process that depends on the combined action of glycogen degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose and maltotetraose from glycogen was demonstrated. Carbohydrate active enzymes were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single domain -amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an -amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the -amylase-pullulanase, and the single domain -amylase were each produced in E. coli. The -amylase domain from the -amylase-pullulanase released maltose, maltotriose and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella -amylase-pullulanase is capable of hydrolyzing -1,4 and -1,6 glycosidic bonds. Similarly, the single domain -amylase protein also produced maltose, maltotriose and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as public goods that can digest glycogen into maltose, maltotriose and maltotetraose that can be used by the vaginal microbiota. ImportanceIncreased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

microbiology↗

bioRxiv2021

Established Gardnerella biofilms can survive metronidazole treatment by reducing metabolic activity

Gardnerella spp. can form biofilm and it has been suggested that failure of antibiotic treatment of bacterial vaginosis and recurrent vaginosis are linked to this property, however no specific mechanisms have been identified. Here, we performed broth microdilution assays to measure the minimum inhibitory concentration (MIC) of metronidazole for thirty-five Gardnerella isolates in one medium in which Gardnerella spp. grow primarily as biofilm and another in which growth is primarily planktonic. The MIC of isolates in the two conditions were highly correlated (R2= 0.69, p <0.001). No significant reduction in viable cell count of 7/9 isolates was observed when established biofilms were exposed to metronidazole at levels double the MIC. Subsequent measurements of metabolic activity in established biofilms formed by a larger collection of 31 isolates showed reduced metabolic activity following treatment with 128 g/ml of metronidazole relative to untreated controls in 27/31 cases. The amount of biofilm produced by Gardnerella isolates was not enhanced by metronidazole exposure, even at sub-MIC levels. Our results suggest that reduced metabolic activity in Gardnerella spp. growing in established biofilms may be a mechanism of protection from the bactericidal effects of metronidazole.

microbiology↗

bioRxiv2021

cpn60 barcode sequences accurately identify newly defined genera within the Lactobacillaceae

The cpn60 barcode sequence is established as an informative target for microbial species identification. Applications of cpn60 barcode sequencing are supported by the availability of "universal" PCR primers for its amplification and a curated reference database of cpn60 sequences, cpnDB. A recent reclassification of lactobacilli involving the definition of 23 new genera provided an opportunity to update cpnDB and to determine if the cpn60 barcode could be used for accurate identification of species consistent with the new framework. Analysis of 275 cpn60 sequences representing 258/269 of the validly named species in Lactobacillus, Paralactobacillus and the 23 newer genera showed that cpn60-based sequence relationships were consistent with the whole-genome-based phylogeny. Aligning or mapping full length barcode sequences or a 150 bp subsequence resulted in accurate and unambiguous species identification in almost all cases. Taken together, our results show that the combination of available reference sequence data, "universal" barcode amplification primers, and the inherent sequence diversity within the cpn60 barcode make it a useful target for the detection and identification of lactobacilli as defined by the latest taxonomic framework. Significance and Impact of the StudyThe genus Lactobacillus recently underwent a major reorganization resulting in the definition of 23 new genera. Lactobacilli are widespread in environmental and host-associated microbiomes and are exploited in food and biotechnology applications, making methods for their accurate identification desirable. Here we show that the combination of a reference sequence database, "universal" barcode amplification primers, and the inherent sequence diversity within the cpn60 barcode make it a useful target for the detection and identification of lactobacilli as defined by the latest taxonomic framework.

microbiology↗

bioRxiv2021

Population density affects the outcome of competition in co-cultures of Gardnerella species isolated from the human vaginal microbiome

Negative frequency-dependent selection is one possible mechanism for maintenance of rare species in communities, but the selective advantage of rare species may be checked at lower overall population densities where resources are abundant. Gardnerella spp. belonging to cpn60 subgroup D, are detected at low levels in vaginal microbiomes and are nutritional generalists relative to other more abundant Gardnerella spp., making them good candidates for negative frequency-dependent selection. The vaginal microbiome is a dynamic environment and the resulting changes in density of the microbiota may explain why subgroup D never gains dominance. To test this, we co-cultured subgroup D isolates with isolates from the more common and abundant subgroup C. Deep amplicon sequencing of rpoB was used to determine proportional abundance of each isolate at 0 h and 72 h in 152 co-cultures, and to calculate change in proportion. D isolates had a positive change in proportional abundance in most co-cultures regardless of initial proportion. Initial density affected the change in proportion of subgroup D isolates either positively or negatively depending on the particular isolates combined, suggesting that growth rate, population density and other intrinsic features of the isolates influenced the outcome. Our results demonstrate that population density is an important factor influencing the outcome of competition between Gardnerella spp. isolated from the human vaginal microbiome.

microbiology↗

bioRxiv2020

Slipped strand mispairing in the gene encoding cell wall associated sialidase NanH3 in Gardnerella spp.

Cell wall proteins with sialidase activity are involved in carbohydrate assimilation, adhesion to mucosal surfaces, and biofilm formation. Gardnerella spp. inhabit the human vaginal microbiome and encode up to three sialidase enzymes, two of which are suspected to be cell wall associated. Here we demonstrate that the gene encoding extracellular sialidase NanH3 is found almost exclusively in G. piotii and closely related Gardnerella genome sp. 3, and its presence correlates with sialidase positive phenotype in a collection of 112 Gardnerella isolates. The nanH3 gene sequence includes a homopolymeric repeat of cytosines that varies in length within cell populations, indicating that this gene is subject to slipped-strand mispairing, a mechanisms of phase variation in bacteria. Variation in the length of the homopolymer sequence results in encoding of either the full length sialidase protein or truncated peptides lacking the sialidase domain due to introduction of reading-frame shifts and premature stop codons. Phase variation in NanH3 may be involved in immune evasion or modulation of adhesion to host epithelial cells, and formation of biofilms characteristic of the vaginal dysbiosis known as bacterial vaginosis.

microbiology↗

bioRxiv2020

A generalist lifestyle allows rare Gardnerella spp. to persist at low levels in the vaginal microbiome

Gardnerella spp. are considered a hallmark of bacterial vaginosis, a dysbiosis of the vaginal microbiome. There are four cpn60 sequence-based subgroups within the genus (A, B, C, and D), and thirteen genome species have been defined recently. Gardnerella spp. co-occur in the vaginal microbiome with varying abundance, and these patterns are shaped by a resource-dependent, exploitative competition, which affects the growth rate of subgroup A, B, and C negatively. The growth rate of rarely abundant subgroup D, however, increases with the increasing number of competitors, negatively affecting the growth rate of others. We hypothesized that a nutritional generalist lifestyle and minimal niche overlap with the other, more abundant Gardnerella spp. facilitate the maintenance of subgroup D in the vaginal microbiome through negative-frequency dependent selection. Using 40 whole genome sequences from isolates representing all four subgroups we found that they could be distinguished based on content of their predicted proteomes. Proteins associated with carbohydrate and amino acid uptake and metabolism were significant contributors to the separation of subgroups. Subgroup D isolates had significantly more of their proteins assigned to amino acid metabolism than the other subgroups. Subgroup D isolates were also significantly different from others in terms of number and type of carbon sources utilized in a phenotypic assay, while the other three could not be distinguished. Overall, the results suggest that a generalist lifestyle and lack of niche overlap with other Gardnerella spp. leads to subgroup D being favoured by negative-frequency dependent selection in the vaginal microbiome.

microbiology↗

bioRxiv2020

Assessment of metagenomic sequencing and qPCR for detection of influenza D virus in bovine respiratory tract samples

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: Whats In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

microbiology↗

bioRxiv2020

Characterization of an α-glucosidase enzyme conserved in Gardnerella spp. isolated from the human vaginal microbiome

Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, a dysbiosis in which a lactobacilli dominant microbial community is replaced with mixed aerobic and anaerobic bacteria including Gardnerella species. The co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, particularly glycogen present in the vaginal environment, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 Gardnerella genomes demonstrated that a putative -glucosidase-encoding sequence, CG400_06090, was conserved in all species in the genus. The predicted -glucosidase enzyme was expressed, purified and functionally characterized. The enzyme was active on a variety of maltooligosaccharides over a broad pH range (4.0 - 8) with maximum activity at pH 7. The Km, kcat and kcat/Km values for the substrate 4-nitrophenyl -D-glucopyranoside were 8.3 M, 0.96 min-1 and 0.11 M-1min-1 respectively. Glucose was released from maltose, maltotriose, maltotetraose and maltopentaose, but no products were detected on thin layer chromatography when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an -glucosidase enzyme that may contribute to the complex and multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides.

microbiology↗

bioRxiv2019

Variant calling for cpn60 barcode sequence-based microbiome profiling

Amplification and sequencing of conserved genetic barcodes such as the cpn60 gene is a common approach to determining the taxonomic composition of microbiomes. Exact sequence variant calling has been proposed as an alternative to previously established methods for aggregation of sequence reads into operational taxonomic units (OTU). We investigated the utility of variant calling for cpn60 barcode sequences and determined the minimum sequence length required to provide species-level resolution. Sequence data from the 5 region of the cpn60 barcode amplified from the human vaginal microbiome (n=45), and a mock community were used to compare variant calling to de novo assembly of reads, and mapping to a reference sequence database in terms of number of OTU formed, and overall community composition. Variant calling resulted in microbiome profiles that were consistent in apparent composition to those generated with the other methods but with significant logistical advantages. Variant calling is rapid, achieves high resolution of taxa, and does not require reference sequence data. Our results further demonstrate that 150 bp from the 5 end of the cpn60 barcode sequence is sufficient to provide species-level resolution of microbiota.

bioinformatics↗

bioRxiv2019

Competition among Gardnerella subgroups from the human vaginal microbiome

Gardnerella spp. are hallmarks of bacterial vaginosis, a clinically significant dysbiosis of the vaginal microbiome. Gardnerella has four subgroups (A, B, C and D) based on cpn60 sequences. Multiple subgroups are often detected in individual women, and interactions between these subgroups are expected to influence their population dynamics and associated signs and symptoms of bacterial vaginosis. In the present study, contact-independent and contact-dependent interactions between the four Gardnerella subgroups were investigated in vitro. The cell free supernatants of mono- and co-cultures had no effect on growth rates of the Gardnerella subgroups suggesting that there are no contact-independent interactions (and no contest competition). For contact-dependent interactions, mixed communities of 2, 3 or 4 subgroups were created and the initial (0 h) and final population sizes (48 h) were quantified using subgroup-specific PCR. Compared to the null hypothesis of additive interactions, most (69.3%) of the mixed communities exhibited competition (p < 0.0001). Competition reduced the growth rates of subgroups A, B and C. In contrast, the growth rate of subgroup D increased in the presence of the other subgroups (p < 0.0001). All subgroups were able to form biofilm alone and in mixed communities. Our study suggests that there is scramble competition among Gardnerella subgroups, which likely contributes to the observed distributions of Gardnerella spp. in vaginal microbiomes and the formation of the multispecies biofilms characteristic of bacterial vaginosis.

microbiology↗

bioRxiv2019

Resolution and co-occurrence patterns of Gardnerella leopoldii, Gardnerella swidsinskii, Gardnerella piotii and Gardnerella vaginalis within the vaginal microbiome

BackgroundGardnerella vaginalis is a hallmark of vaginal dysbiosis, but is found in the microbiomes of women with and without vaginal symptoms. G. vaginalis encompasses diverse taxa differing in attributes that are potentially important for virulence, and there is evidence that clades or subgroups within the species are differentially associated with clinical outcomes. The G. vaginalis species description was recently emended, and three new species within the genus were defined (leopoldii, swidsinskii, piotii). 16S rRNA sequences for the four Gardnerella species are all >98.5% identical and no signature sequences differentiate them.\n\nResultsWe demonstrated that Gardnerella species can be resolved using partial chaperonin-60 (cpn60) sequences, with pairwise percent identities of 87.1-97.8% among the type strains. Pairwise co-occurrence patterns of Gardnerella spp. in the vaginal microbiomes of 413 reproductive aged Canadian women were investigated, and several significant co-occurrences of species were identified. Abundance of G. vaginalis, and swidsinskii was associated with vaginal symptoms of abnormal odour and discharge.\n\nConclusionscpn60 barcode sequencing can provide a rapid assessment of the relative abundance of Gardnerella spp. in microbiome samples, providing a powerful method of elucidating associations between these diverse organisms and clinical outcomes. Researchers should consider using cpn60 in place of 16S RNA for better resolution of these important organisms.

microbiology↗

bioRxiv2019

Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2

Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces arsenal of signalling and antagonistic compounds.

microbiology↗