Grand Challenges in Microbiotechnology: Through the Prism of Microbiotechnology.

Microbes have conquered almost every conceivable space on earth—from high atmospheres to terrestrial and aquatic ecosystems to extreme places such as geothermal vents in the deep sea, oil reservoirs, or boiling hot springs. Survival in these varied environments necessitates a breathtaking span of genetic diversity, enabling the metabolism and synthesis of many different substrates for both energy creation and biomass buildup and to gain an evolutionary advantage over other life forms sharing the same ecosystem. Of particular biotechnological interest are molecules referred to as secondary metabolites that often feature a unique chemical makeup and can encompass functions such as ion scavenging, quorum sensing, or act as antimicrobials.fals

Genome sequence of the entomopathogenic Serratia entomophila isolate 626 and characterisation of the species specific itaconate degradation pathway

BACKGROUND: Isolates of Serratia entomophila and S. proteamaculans (Yersiniaceae) cause disease specific to the endemic New Zealand pasture pest, Costelytra giveni (Coleoptera: Scarabaeidae). Previous genomic profiling has shown that S. entomophila isolates appear to have conserved genomes and, where present, conserved plasmids. In the absence of C. giveni larvae, S. entomophila prevalence reduces in the soil over time, suggesting that S. entomophila has formed a host-specific relationship with C. giveni. To help define potential genetic mechanisms driving retention of the chronic disease of S. entomophila, the genome of the isolate 626 was sequenced, enabling the identification of unique chromosomal properties, and defining the gain/loss of accessory virulence factors relevant to pathogenicity to C. giveni larvae. RESULTS: We report the complete sequence of S. entomophila isolate 626, a causal agent of amber disease in C. giveni larvae. The genome of S. entomophila 626 is 5,046,461 bp, with 59.1% G + C content and encoding 4,695 predicted CDS. Comparative analysis with five previously sequenced Serratia species, S. proteamaculans 336X, S. marcescens Db11, S. nematodiphila DH-S01, S. grimesii BXF1, and S. ficaria NBRC 102596, revealed a core of 1,165 genes shared. Further comparisons between S. entomophila 626 and S. proteamaculans 336X revealed fewer predicted phage-like regions and genomic islands in 626, suggesting less horizontally acquired genetic material. Genomic analyses revealed the presence of a four-gene itaconate operon, sharing a similar gene order as the Yersinia pestis ripABC complex. Assessment of a constructed 626::RipC mutant revealed that the operon confer a possible metabolic advantage to S. entomophila in the initial stages of C. giveni infection. CONCLUSIONS: Evidence is presented where, relative to S. proteamaculans 336X, S. entomophila 626 encodes fewer genomic islands and phages, alluding to limited horizontal gene transfer in S. entomophila. Bioassay assessments of a S. entomophila-mutant with a targeted mutation of the itaconate degradation region unique to this species, found the mutant to have a reduced capacity to replicate post challenge of the C. giveni larval host, implicating the itaconate operon in establishment within the host.fals

Draft Genome Sequences of the Type Strains of Six Macrococcus Species.

We report here the draft genome sequences of Macrococcus bovicus ATCC 51825T, Macrococcus carouselicus ATCC 51828T, Macrococcus equipercicus ATCC 51831T, Macrococcus brunensis CCM4811T, Macrococcus hajekii CCM4809T, and Macrococcus lamae CCM4815T The availability of the genome sequences of these species will enable cross-species comparison, which could lead to a more comprehensive understanding of organisms of the Macrococcus genus.fals

PLAN-M; Mycobacteriophage Endolysins Fused to Biodegradable Nanobeads Mitigate Mycobacterial Growth in Liquid and on Surfaces.

The Mycobacteria are a genus of Actinobacteria that include human pathogens such as Mycobacterium tuberculosis (TB). Active TB disease can spread by airborne transmission to healthcare workers and to their community. The HHMI SEA-PHAGES program has contributed to discovering bacteriophages that are able to infect M. smegmatis MC2 155, a close relative of M. tuberculosis. This collection of diverse Mycobacteriophages is an excellent resource for trialling bacteriophage-sourced enzymes in novel applications. Herein we measured the ability Mycobacteriophage endolysins to lyse their host strain when functionally fused to biodegradable polyhydroxyalkanoate (PHA) nanobeads. PHA nanobeads facilitate both the expression and the application of enzymes to surfaces and have been demonstrated to stabilize a wide array of proteins for practical applications whilst eliminating the challenges of traditional protein purification. We selected two Lysin A and six Lysin B homologs to be functionally fused to the polyhydroxyalkanoate synthase C (PhaC). Expression of these constructs resulted in functional lysins displayed on the surface of PHA nanobeads. The lysins thus directionally displayed on nanobeads lysed up to 79% of the M. smegmatis MC2 155 population using 80 mg/mL of nanobeads in pure culture. In order to determine whether the nanobeads would be effective as a protective layer in PPE we adapted a fabric-based test and observed a maximum of 1 log loss of the cell population after 5 h of exposure on a textile (91% cell lysis). Lysin B enzymes performed better than the Lysin A enzymes as a protective barrier on textiles surface assays. These results suggest that bacterial endolysins are efficient in their action when displayed on PHA nanobeads and can cause significant population mortality in as little as 45 min. Our results provide the proof-of-principle that Mycobacteriophage endolysins can be used on functionalized nanobeads where they can protect surfaces such as personal protective equipment (PPE) that routinely come into contact with aerosolised bacteria.fals

Draft Genome Sequences of Macrococcus caseolyticus, Macrococcus canis, Macrococcus bohemicus, and Macrococcus goetzii.

Here, we present the draft genome sequences of 14 strains of 4 species of the genus Macrococcus These strains were isolated from bovine milk and tongue samples obtained during a screening program.fals

The Effects of Unfermented and Fermented Cow and Sheep Milk on the Gut Microbiota.

A variety of fermented foods have been linked to improved human health, but their impacts on the gut microbiome have not been well characterized. Dairy products are one of the most popular fermented foods and are commonly consumed worldwide. One area we currently lack data on is how the process of fermentation changes the gut microbiota upon digestion. What is even less well characterized are the possible differences between cow and other mammals' milks. Our aim was to compare the impact of unfermented skim milk and fermented skim milk products (milk/yogurt) originating from two species (cow/sheep) on the gut microbiome using a rat model. Male Sprague-Dawley rats were fed a dairy-free diet supplemented with one of four treatment dairy drinks (cow milk, cow yogurt, sheep milk, sheep yogurt) for 2 weeks. The viable starter culture bacteria in the yogurts were depleted in this study to reduce their potential influence on gut bacterial communities. At the end of the study, cecal samples were collected and the bacterial community profiles determined via 16S rRNA high-throughput sequencing. Fermentation status drove the composition of the bacterial communities to a greater extent than their animal origin. While overall community alpha diversity did not change among treatment groups, the abundance of a number of taxa differed. The cow milk supplemented treatment group was distinct, with a higher intragroup variability and a distinctive taxonomic composition. Collinsella aerofaciens was of particularly high abundance (9%) for this group. Taxa such as Firmicutes and Lactobacillus were found in higher abundance in communities of rats fed with milk, while Proteobacteria, Bacteroidetes, and Parabacteroides were higher in yogurt fed rats. Collinsella was also found to be of higher abundance in both milk (vs. yogurt) and cows (vs. sheep). This research provides new insight into the effects of unfermented vs. fermented milk (yogurt) and animal origin on gut microbial composition in a healthy host. A number of differences in taxonomic abundance between treatment groups were observed. Most were associated with the effects of fermentation, but others the origin species, or in the case of cow milk, unique to the treatment group. Future studies focusing on understanding microbial metabolism and interactions, should help unravel what drives these differences.fals

A Mathematical Model for the Hydrogenotrophic Metabolism of Sulphate-Reducing Bacteria.

Sulphate-reducing bacteria (SRB) are studied across a range of scientific fields due to their characteristic ability to metabolise sulphate and produce hydrogen sulphide, which can lead to significant consequences for human activities. Importantly, they are members of the human gastrointestinal microbial population, contributing to the metabolism of dietary and host secreted molecules found in this environment. The role of the microbiota in host digestion is well studied, but the full role of SRB in this process has not been established. Moreover, from a human health perspective, SRB have been implicated in a number of functional gastrointestinal disorders such as Irritable Bowel Syndrome and the development of colorectal cancer. To assist with the study of SRB, we present a mathematical model for the growth and metabolism of the well-studied SRB, Desulfovibrio vulgaris in a closed system. Previous attempts to model SRB have resulted in complex or highly specific models that are not easily adapted to the study of SRB in different environments, such as the gastrointestinal tract. We propose a simpler, Monod-based model that allows for easy alteration of both key parameter values and the governing equations to enable model adaptation. To prevent any incorrect assumptions about the nature of SRB metabolic pathways, we structure the model to consider only the concentrations of initial and final metabolites in a pathway, which circumvents the current uncertainty around hydrogen cycling by SRB. We parameterise our model using experiments with varied initial substrate conditions, obtaining parameter values that compare well with experimental estimates in the literature. We then validate our model against four independent experiments involving D. vulgaris with further variations to substrate availability. Further use of the model will be possible in a number of settings, notably as part of larger models studying the metabolic interactions between SRB and other hydrogenotrophic microbes in the human gastrointestinal tract and how this relates to functional disorders.fals

The Role of Segmented Filamentous Bacteria in Immune Barrier Maturation of the Small Intestine at Weaning.

The microbiological, physical, chemical, and immunological barriers of the gastrointestinal tract (GIT) begin developing in utero and finish maturing postnatally. Maturation of these barriers is essential for the proper functioning of the GIT. Maturation, particularly of the immunological barrier, involves stimulation by bacteria. Segmented filamentous bacteria (SFB) which are anaerobic, spore-forming commensals have been linked to immune activation. The presence and changes in SFB abundance have been positively correlated to immune markers (cytokines and immunoglobulins) in the rat ileum and stool samples, pre- and post-weaning. The abundance of SFB in infant stool increases from 6 months, peaks around 12 months and plateaus 25 months post-weaning. Changes in SFB abundance at these times correlate positively and negatively with the production of interleukin 17 (IL 17) and immunoglobulin A (IgA), respectively, indicating involvement in immune function and maturation. Additionally, the peak in SFB abundance when a human milk diet was complemented by solid foods hints at a diet effect. SFB genome analysis revealed enzymes involved in metabolic pathways for survival, growth and development, host mucosal attachment and substrate acquisition. This narrative review discusses the current knowledge of SFB and their suggested effects on the small intestine immune system. Referencing the published genomes of rat and mouse SFB, the use of food substrates to modulate SFB abundance is proposed while considering their effects on other microbes. Changes in the immune response caused by the interaction of food substrate with SFB may provide insight into their role in infant immunological barrier maturation.fals

Mathematical modelling supports the existence of a threshold hydrogen concentration and media-dependent yields in the growth of a reductive acetogen.

The bacterial production of acetate via reductive acetogenesis along the Wood-Ljungdahl metabolic pathway is an important source of this molecule in several environments, ranging from industrial bioreactors to the human gastrointestinal tract. Here, we contributed to the study of reductive acetogens by considering mathematical modelling techniques for the prediction of bacterial growth and acetate production. We found that the incorporation of a hydrogen uptake concentration threshold into the models improves their predictions and we calculated this threshold as 86.2 mM (95% confidence interval 6.1-132.6 mM). Monod kinetics and first-order kinetics models, with the inclusion of two candidate threshold terms or reversible Michaelis-Menten kinetics, were compared to experimental data and the optimal formulation for predicting both growth and metabolism was found. The models were then used to compare the efficacy of two growth media for acetogens. We found that the recently described general acetogen medium was superior to the DSMZ medium in terms of unbiased estimation of acetogen growth and investigated the contribution of yeast extract concentration to acetate production and bacterial growth in culture. The models and their predictions will be useful to those studying both industrially and environmentally relevant reductive acetogenesis and allow for straightforward adaptation to similar cases with different organisms.fals

Exploring rumen microbe-derived fibre-degrading activities for improving feed digestibility

Ruminal fibre degradation is mediated by a complex community of rumen microbes, and its efficiency is crucial for optimal dairy productivity. Enzymes produced by rumen microbes are primarily responsible for degrading the complex structural polysaccharides that comprise fibre in the plant cell walls of feed materials. Because rumen microbes have evolved with their ruminant hosts over millions of years to perform this task, their enzymes are hypothesised to be optimally suited for activity at the temperature, pH range, and anaerobic environment of the rumen. However, fibre-rich diets are not fully digested, which represents a loss in potential animal productivity. Thus, there is opportunity to improve fibre utilisation through treating feeds with rumen microbe-derived fibrolytic enzymes and associated activities that enhance fibre degradation. This research aims to gain a better understanding of the key rumen microbes involved in fibre degradation and the mechanisms they employ to degrade fibre, by applying cultivation-based and culture-independent genomics approaches to rumen microbial communities of New Zealand dairy cattle. Using this knowledge, we aim to identify new opportunities for improving fibre degradation to enhance dairy productivity. Rumen content samples were taken over the course of a year from a Waikato dairy production herd. Over 1,000 rumen bacterial cultures were obtained from the plant-adherent fraction of the rumen contents. Among these cultures, two, 59 and 103 potentially new families, genera and species of rumen bacteria were identified, respectively. Many of the novel strains are being genome sequenced within the Hungate 1000 rumen microbial reference genome programme, which is providing deeper insights into the range of mechanisms used by the individual strains for fibre degradation. This information has been used to guide the selection of rumen bacterial strains with considerable potential as fibrolytic enzyme producers in vitro, with the intent of developing the strains so that their enzymes may be used as feed pre-treatments for use on farm. Culture-independent metagenomic approaches were also used to explore the activities involved in fibre degradation from the rumen microbial communities. Functional screening has revealed a range of novel enzymes and a novel fibre disrupting activity. Enrichment for the cell-secreted proteins from the community revealed evidence of a diverse range of cellulosomes, which are cell-surface associated multi-enzyme complexes that efficiently degrade plant cell wall polysaccharides. Biochemical and structural characterisation of these proteins has been conducted. In conclusion, cultivation and culture-independent genomic approaches have been applied to New Zealand bovine rumen microbial communities, and have provided considerable new insights into ruminal fibre degradation processes. Novel activities and bacterial species that display desirable activities on fibrous substrates in vitro are now being explored for their potential to improve ruminal fibre degradation, to allow the development of new technologies that will enhance dairy productivity