Applied and Environmental Microbiology最新文献

The Pseudomonas putida F1 genome and those of many other pseudomonads contain two tandem genes encoding acyl-CoA ligases Pput_1340 (fadD1) and Pput_1339 (fadD2) with Pput_1339 (fadD2) being the upstream gene. The fadD designation was assigned when both genes were found to complement the growth of an Escherichia coli acyl-CoA synthetase fadD deletion strain with oleic acid as sole carbon source. Site-directed mutagenesis showed that residues of the ATP/AMP domain required for function of E. coli FadD were also essential for full function of FadD1 and FadD2. Growth of the constructed ∆fadD1, ∆fadD2, and ∆fadD1∆fadD2 strains was tested in minimal medium with different chain length fatty acids as sole carbon sources. Lack of FadD1 significantly retarded growth with different chain length fatty acids and lack of both FadD1 and FadD2 further retarded growth. Derivatives of the ∆fabA∆desA unsaturated fatty acid auxotrophic strain carrying a deletion of either ∆fadD1 or ∆fadD2 were constructed. Growth of the ∆fabA∆desA∆fadD1 strain was very weak, whereas the ∆fabA∆desA∆fadD2 strain grew as well as the ∆fabA∆desA parent strain. Overexpression of either fadD1 or fadD2 restored growth of the ∆fabA∆desA∆fadD1 strain with fadD2 overexpression having a greater effect than fadD1 overexpression. The ∆fadD1 or ∆fadD2 genes are cotranscribed although the expression level of fadD1 is much higher than that of fadD2. This is attributed to a fadD1 promoter located within the upstream FadD2 coding sequence.

Importance: Pseudomonas bacteria demonstrate a great deal of metabolic diversity and consequently colonize a wide range of ecological niches. A characteristic of these bacteria is a pair of genes in tandem annotated as acyl-CoA ligases involved in fatty acid degradation. The Pseudomonas putida F1 genome is annotated as having at least nine genes encoding acyl-CoA ligases which are scattered around the chromosome excepting the tandem pair. Since similar tandem pairs are found in other pseudomonads, we have constructed and characterized deletion mutants of the tandem ligases. We report that the encoded proteins are authentic acyl-CoA ligases involved in fatty acid degradation.

Black soldier fly larvae (BSFL) have attracted attention due to their ability to upcycle various biological side streams into valuable biomass, such as proteins, lipids, and chitin. In this study, we investigated the impact of high-fiber diets on larval growth performance and the shift of microbes in the gut. We tested empty fruit bunches (EFB), potato pulp (PP), and cottonseed press cake (CPC), with chicken feed (CF) used as a control diet. We found that larvae reared on the EFB, PP, and CPC were smaller than control larvae at the end of development due to the low nutritional value of the diets. However, survival rates of more than 90% were observed regardless of the diet. We used a cultivation-dependent approach to analyze the microbial community in the gut of BSFL, isolated, and identified a total of 329 bacterial strains. Bacillaceae were most frequently isolated from larvae reared on the high-fiber EFB diet. These isolates were predicted to degrade cellulose in silico and this was subsequently confirmed in vitro using the Congo Red assay. Whereas the members of Enterobacteriaceae and Morganellaceae were mostly found in guts of larvae reared on the high-protein diets CPC and CF. We conclude that the gut microbiome plays a crucial role in the digestion of fiber-rich plant organic material, thereby enabling the BSFL to successfully complete their life cycle also on substrates with low nutritional value. As a result, BSFL convert industrial side streams into valuable biomass, reducing waste and promoting sustainability.

Importance: Organic side streams from various industries pose a challenge to the environment. They are often present in huge amounts and are mostly discarded, incinerated, used for biogas production, or as feed for ruminant animals. Many plant-based side streams contain difficult-to-digest fiber as well as anti-nutritional or even insecticidal compounds that could harm the animals. These challenges can be addressed using black soldier fly larvae, which are known to degrade various organic substrates and convert them into valuable biomass. This will help mitigate agro-industrial side streams via efficient waste management and will contribute to the more economical and sustainable farming of insects.

Bacterial biotherapeutic delivery vehicles have the potential to treat a variety of diseases. This approach obviates the need to purify the recombinant effector molecule, allows delivery of therapeutics in situ via oral or intranasal administration, and protects the effector molecule during gastrointestinal transit. Lactic acid bacteria have been broadly developed as therapeutic delivery vehicles though risks associated with the colonization of a genetically modified microorganism have so-far not been addressed. Here, we present an engineered Limosilactobacillus reuteri strain with reduced colonization potential. We applied a dual-recombineering scheme for efficient barcoding and generated mutants in genes encoding five previously characterized and four uncharacterized putative adhesins. Compared with the wild type, none of the mutants were reduced in their ability to survive gastrointestinal transit in mice. CmbA was identified as a key protein in L. reuteri adhesion to HT-29 and enteroid cells. The nonuple mutant, a single strain with all nine genes encoding adhesins inactivated, had reduced capacity to adhere to enteroid monolayers. The nonuple mutant producing murine IFN-β was equally effective as its wild-type counterpart in mitigating radiation toxicity in mice. Thus, this work established a novel therapeutic delivery platform that lays a foundation for its application in other microbial therapeutic delivery candidates and furthers the progress of the L. reuteri delivery system towards human use.IMPORTANCEOne major advantage to leverage gut microbes that have co-evolved with the vertebrate host is that evolution already has taken care of the difficult task to optimize survival within a complex ecosystem. The availability of the ecological niche will support colonization. However, long-term colonization of a recombinant microbe may not be desirable. Therefore, strategies need to be developed to overcome this potential safety concern. In this work, we developed a single strain in which we inactivated the encoding sortase, and eight genes encoding characterized/putative adhesins. Each individual mutant was characterized for growth and adhesion to epithelial cells. On enteroid cells, the nonuple mutant has a reduced adhesion potential compared with the wild-type strain. In a model of total-body irradiation, the nonuple strain engineered to release murine interferon-β performed comparable to a derivative of the wild-type strain that releases interferon-β. This work is an important step toward the application of recombinant L. reuteri in humans.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage.IMPORTANCEDirected evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies.

Human norovirus (HuNoV) is recognized as the leading causative agent of foodborne outbreaks of epidemic gastroenteritis. Consequently, there is a high demand for developing point-of-care testing for HuNoV. We developed an origami microfluidic device that facilitates rapid detection of murine norovirus 1 (MNV-1), a surrogate for HuNoV, encompassing the entire process from sample preparation to result visualization. This process includes RNA absorption via a paper strip, RNA amplification using recombinase polymerase amplification (RPA), and a lateral flow assay for signal readout. The on-chip detection of MNV-1 was completed within 35 min, demonstrating 100% specificity to MNV-1 in our settings. The detection limit of this microfluidic device for MNV-1 was 200 PFU/mL, comparable to the in-tube RPA reaction. It also successfully detected MNV-1 in lettuce and raspberries at concentrations of 170 PFU/g and 230 PFU/g, respectively, without requiring extra concentration steps. This device demonstrates high compatibility with isothermal nucleic acid amplification and holds significant potential for detecting foodborne viruses in agri-food products in remote and resource-limited settings.

Importance: HuNoV belongs to the family of Caliciviridae and is a leading cause of acute gastroenteritis that can be transmitted through contaminated foods. HuNoV causes around one out of five cases of acute gastroenteritis that lead to diarrhea and vomiting, placing a substantial burden on the healthcare system worldwide. HuNoV outbreaks can occur when food is contaminated at the source (e.g., wild mussels exposed to polluted water), on farms (e.g., during crop cultivation, harvesting, or livestock handling), during packaging, or at catered events. The research outcomes of this study expand the approaches of HuNoV testing, adding value to the framework for routine testing of food products. This microfluidic device can facilitate the monitoring of HuNoV outbreaks, reduce the economic loss of the agri-food industry, and enhance food safety.

Dietary fibers play a crucial role in shaping the gut microbiome and influencing gastrointestinal (GI) physiology. Grain-based diets (GBDs) are widely used in rodent studies, but their utility is limited due to batch-to-batch variability resulting from inconsistent ingredients. Purified diets (PDs) are composed of only known and refined ingredients and offer a solution to the constraints of GBDs. This study aimed to identify a combination of dietary fibers in a purified diet (PD) that promotes optimal murine gut morphometry and a diverse intestinal microbial community. Male C57BL/6J mice were fed either two grain-based diets (GBDs) or four PDs with varying fiber compositions for 28 days. Mice consuming PDs lacking soluble fiber had more gonadal fat (P < 0.05), shorter small intestines (P < 0.05), and lighter ceca (P < 0.05) compared with those fed the LabDiet 5001 GBD. Increasing the proportion of soluble fibers in PDs progressively reduced microbial diversity in the cecum and colon. Multidimensional scaling analysis revealed distinct microbial communities in the cecum and colon between mice fed GBDs and PDs (P < 0.05). Differential abundance analysis identified relatively more Family XII UCG 001 and less Lactococcus in mice fed GBDs relative to mice consuming PDs (P < 0.05). While no PD recapitulated the gut microbial composition of GBDs, PDs with high soluble fiber content best preserved GI morphometry. These findings underscore the importance of considering diet as an experimental variable and highlight the need for a PD formulation that combines the benefits of GBDs on GI health and microbial richness.

Importance: Dietary fibers are essential for maintaining gut health. Insoluble fibers aid in fecal bulking and water retention while soluble fiber is a fermentative substrate for intestinal microbial communities. Grain-based diets (GBDs) are commonly used in preclinical research but the variability in ingredients across batches impedes reproducibility. Purified diets (PDs), which are composed of highly refined ingredients, pose a potential solution but the most widely used low-fat control PDs contain no soluble fiber. This study intended to identify a PD with a combination of fibers that promotes murine gut health and microbial diversity. A PD with optimal fiber composition would aid in the standardization and reproducibility of studies investigating intestinal physiology and the gut microbiota.

The rise of the Isthmus of Panama separated the populations of many marine organisms, which then diverged into new geminate sister species currently living in the Eastern Pacific Ocean and the Caribbean Sea. However, we know very little about how such evolutionary divergences of host species have shaped the compositions of their microbiomes. Here, we compared the microbiomes of whole-body and shell-surface samples of geminate species of marine gastropods in the genera Cerithium and Cerithideopsis to those of congeneric outgroups. Our results suggest that the effects of ~3 million years of separation and isolation on microbiome composition varied among host genera and between sample types within the same hosts. In the whole-body samples, microbiome compositions of geminate species pairs tended to be similar, likely due to host filtering, although the strength of this relationship varied among the two groups and across similarity metrics. Shell-surface microbiomes show contrasting patterns, with co-divergence between the host taxa and a small number of microbial clades evident in Cerithideopsis but not Cerithium. These results suggest that (i) isolation of host populations after the rise of the Isthmus of Panama affected microbiomes of geminate hosts in a complex and host-specific manner, and (ii) host-associated microbial taxa respond differently to vicariance events than the hosts themselves.IMPORTANCEWhile considerable work has been done on evolutionary divergences of marine species in response to the rise of the Isthmus of Panama, which separated two previously connected oceans, how this event shaped the microbiomes of these marine hosts remains poorly known. Using whole-body and shell-surface microbiomes of closely related gastropod species from opposite sides of the Isthmus, we show that divergences of microbial taxa after the formation of the Isthmus are often not concordant with those of their gastropod hosts. Our results show that evolutionary responses of marine gastropod-associated microbiomes to major environmental perturbations are complex and are shaped more by local environments than host evolutionary history.

In this study, the effect of sodium hypochlorite (10-200 ppm of Cl₂) on the inactivation of human norovirus (HuNV) GII.4 and hepatitis A virus (HAV) in groundwater was investigated using propidium monoazide (PMA)/reverse transcription quantitative real-time PCR (RT-qPCR). Initially, 4.00 log₁₀ genome copies/μL of HuNV GII.4 or 5.50 log₁₀ genome copies/μL of HAV were artificially inoculated in groundwater. The titers of HuNV GII.4 and HAV decreased significantly (P < 0.05) with increasing Cl₂ concentrations. Groundwater was treated with 10, 30, 50, 100, 150, and 200 ppm of Cl₂, and the viable HuNV GII.4 was significantly (P < 0.05) reduced to 3.28 (0.21-log reduction), 3.18 (0.31-log reduction), 3.01 (0.48 log reduction), 2.75 (0.74 log reduction), 2.54 (0.95 log reduction), and 2.34 (1.15 log reduction) log₁₀ genome copies/μL, respectively. The viable HAV was also significantly (P < 0.05) reduced to 4.99 (0.23 log reduction), 4.76 (0.46 log reduction), 4.55 (0.67 log reduction), 4.21 (1.01-log reduction), 3.89 (1.33 log reduction), and 3.64 (1.58 log reduction) log₁₀ genome copies/μL, respectively. The decimal reduction times (D values) (1-log₁₀ genome reduction) of HuNV GII.4 and HAV infectivity in groundwater were predicted as 116.7 and 98.9 ppm of Cl₂, respectively, using the first-order kinetics model (HuNV GII.4: y = -0.0054x + 3.3585, correlation coefficient (R²) = 0.97; HAV: y = -0.0091x + 5.0470, R² = 0.97). The result specifically suggests that 150- to 200-ppm Cl₂ can potentially be used for the inactivation of >1-log₁₀ genome copy/μL HuNV GII.4 and HAV in groundwater.IMPORTANCEGroundwater represents a vital component of the global water supply, serving as a crucial source of potable water for humans. It serves as a source of potable water for up to 50% of the global population and accounts for 43% of all water used for irrigation. It thus follows that the sustainable management of groundwater represents a pivotal solution. However, the regrowth of pathogens in water that is not treated with chlorine or where proper residual chlorine is not maintained represents a risk to public health.

Molecular efflux is a mechanism through which bacteria actively expel undesirable substances. This is a crucial line of defense against toxic chemicals in harsh environments. Understanding how efflux works is critical for designing antimicrobial strategies. Though much is already known about efflux proteins, important details about the mechanisms of efflux (e.g., importance of specific subcellular domains and ejection rates) have yet to be experimentally quantified. Herein, we use the nonlinear optical technique, second harmonic light scattering, to simultaneously measure the efflux rates from the periplasm and cytosol of a Gram-negative bacterium. The influence of efflux on the uptake kinetics of a mild antimicrobial, malachite green (MG), by Pseudomonas aeruginosa was quantified. It is observed that efflux primarily occurs from the periplasm and is two orders of magnitude faster than from the cytosol. Efflux pumps activate to maintain MG concentrations in the periplasm below 1 µM, while efflux from the cytosol maintains MG concentration below 0.1 µM. Efflux pumps are shown to saturate when exogenous MG concentrations are greater than 25 µM, while the cytosol efflux function saturates at >15 µM. Finally, efflux pumps can simultaneously eject different compounds, as proven by experiments with both MG and hexane, a known effluxable compound.IMPORTANCEMolecular efflux pumps are a crucial defense mechanism that protects bacteria from an otherwise unchecked influx of toxic molecules present in the extracellular environment. The efflux functions constitute a significant hindrance to antimicrobial efficacy. While much is now known regarding the structure of these channels, knowledge of the influence of efflux in individual subcellular domains and the associated ejection rates is still lacking. Using the nonlinear optical technique, second-harmonic light scattering, we have measured the threshold concentrations for pump activation, saturation concentrations, and efflux rates from both the periplasm and cytosol in living Gram-negative bacteria. The quantified efflux data in the different subcellular compartments not only provide a clear mechanistic understanding but also are critical for developing antimicrobial strategies.