Allosteric regulation of menaquinone (vitamin K2) biosynthesis in the human pathogen Mycobacterium tuberculosis.

Menaquinone (vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb). Although menaquinone levels are known to be tightly linked to the cellular redox/energy status of the cell, the regulatory mechanisms underpinning this phenomenon are unclear. The first committed step in menaquinone biosynthesis is catalyzed by MenD, a thiamine diphosphate-dependent enzyme comprising three domains. Domains I and III form the MenD active site, but no function has yet been ascribed to domain II. Here, we show that the last cytosolic metabolite in the menaquinone biosynthesis pathway, 1,4-dihydroxy-2-naphthoic acid (DHNA), binds to domain II of Mtb-MenD and inhibits its activity. Using X-ray crystallography of four apo- and cofactor-bound Mtb-MenD structures, along with several spectroscopy assays, we identified three arginine residues (Arg-97, Arg-277, and Arg-303) that are important for both enzyme activity and the feedback inhibition by DHNA. Among these residues, Arg-277 appeared to be particularly important for signal propagation from the allosteric site to the active site. This is the first evidence of feedback regulation of the menaquinone biosynthesis pathway in bacteria, identifying a protein-level regulatory mechanism that controls menaquinone levels within the cell and may therefore represent a good target for disrupting menaquinone biosynthesis in M. tuberculosis.

Overexpressing ovotransferrin and avian ß-defensin-3 improves antimicrobial capacity of chickens and poultry products.

Zoonotic and foodborne diseases pose a significant burden, decreasing both human and animal health. Modifying chickens to overexpress antimicrobials has the potential to decrease bacterial growth on poultry products and boost chicken innate immunity. Chickens overexpressing either ovotransferrin or avian ß-defensin-3 (AvßD3) were generated using Tol-2 transposons. Transgene expression at the RNA and protein level was seen in egg white, breast muscle, and serum. There were significant differences in the immune cell populations in the blood, bursa, and spleen associated with transgene expression including an increased proportion of CD8+ cells in the blood of ovotransferrin and AvßD3 transgenic birds. Expression of the antimicrobials inhibited the in vitro growth of human and chicken bacterial pathogens and spoilage bacteria. For example, transgene expression significantly reduced growth of aerobic and coliform bacteria in breast muscle and decreased the growth of Salmonella enterica in egg white. Overall these results indicate that overexpression of antimicrobials in the chicken can impact the immune system and increase the antimicrobial capacity of poultry products.

Insight into the Genome of Brochothrix thermosphacta, a Problematic Meat Spoilage Bacterium.

Brochothrix thermosphacta is a dominant but poorly studied meat spoilage organism. It is a close relative of the foodborne pathogen Listeria monocytogenes, and Brochothrix constitutes the second genus in the Listeriaceae family. Here, the genomes of 12 B. thermosphacta strains were sequenced, assembled into draft genomes, characterized, and compared with the genomes of Brochothrix campestris and L. monocytogenes Phenotypic properties including biogenic amine production and antibiotic and heavy metal susceptibilities were tested. Comparative genomic analyses revealed a high degree of similarity among the B. thermosphacta strains, with bacteriophage genes constituting a significant proportion of the accessory genome. Genes for the production of the malodorous compounds acetate, acetoin, butanediol, and fatty acids were found, as were stress response regulatory genes, which likely play important roles in the spoilage process. Amino acid decarboxylases were not identified in the genomes, and phenotypic testing confirmed their absence. Orthologs of Listeria virulence proteins involved in virulence regulation, intracellular survival, and surface protein anchoring were found; however, key virulence genes were absent. Analysis of antibiotic susceptibility showed that strains were sensitive to the four tested antibiotics, except for one tetracycline-resistant isolate with plasmid-mediated tetracycline resistance genes. Strains tolerated higher levels of copper and cobalt than of cadmium although not at concentrations high enough to categorize the strains as being resistant. This study provides insight into the Brochothrix genome, links previous phenotypic data and data provided here to the gene inventory, and identifies genes that may contribute to the persistence of this organism in the food chain.IMPORTANCE Despite increasing knowledge and advances in food preservation techniques, microbial spoilage of foods causes substantial losses, with negative social and economic consequences. To better control the contamination and microbial spoilage of foods, fundamental knowledge of the biology of key spoilage bacteria is crucial. As a common meat spoilage organism, B. thermosphacta contributes substantially to spoilage-associated losses. Nonetheless, this organism and particularly its genome remain largely unstudied. This study contributes to improving our knowledge of the Brochothrix genus. Spoilage-relevant pathways and genes that may play a role in the survival of this organism in a food processing environment were identified, linking previous phenotypic data and data provided here to the gene inventory of Brochothrix and establishing parallels to and differences from the closely related foodborne pathogen L. monocytogenes.

Yar1 protects the ribosomal protein Rps3 from aggregation.

2000 ribosomes have to be synthesized in yeast every minute. Therefore the fast production of ribosomal proteins, their efficient delivery to the nucleus and correct incorporation into ribosomal subunits are prerequisites for optimal growth rates. Here, we report that the ankyrin repeat protein Yar1 directly interacts with the small ribosomal subunit protein Rps3 and accompanies newly synthesized Rps3 from the cytoplasm into the nucleus where Rps3 is assembled into pre-ribosomal subunits. A yar1 deletion strain displays a similar phenotype as an rps3 mutant strain, showing an accumulation of 20S pre-rRNA and a 40S export defect. The combination of an rps3 mutation with a yar1 deletion leads to an enhancement of these phenotypes, while increased expression of RPS3 suppresses the defects of a yar1 deletion strain. We further show that Yar1 protects Rps3 from aggregation in vitro and increases its solubility in vivo. Our data suggest that Yar1 is a specific chaperone for Rps3, which serves to keep Rps3 soluble until its incorporation into the pre-ribosome.

Allosteric inhibition of Staphylococcus aureus MenD by 1,4-dihydroxy naphthoic acid: a feedback inhibition mechanism of the menaquinone biosynthesis pathway.

Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus (Sau), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also use MK, we investigated whether the Sau-MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits the SEPHCHC synthase activity of Sau-MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we showed that exogenous DHNA inhibits the growth of Sau, an effect that can be rescued by supplementing the growth medium with MK-4. Our results demonstrate that, despite a lack of strict conservation of the DHNA binding pocket between Mtb-MenD and Sau-MenD, feedback inhibition by DHNA is a conserved mechanism in Sau-MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Optical Detection of CoV-SARS-2 Viral Proteins to Sub-Picomolar Concentrations.

The emergence of a new strain of coronavirus in late 2019, SARS-CoV-2, led to a global pandemic in 2020. This may have been preventable if large scale, rapid diagnosis of active cases had been possible, and this has highlighted the need for more effective and efficient ways of detecting and managing viral infections. In this work, we investigate three different optical techniques for quantifying the binding of recombinant SARS-CoV-2 spike protein to surface-immobilized oligonucleotide aptamers. Biolayer interferometry is a relatively cheap, robust, and rapid method that only requires very small sample volumes. However, its detection limit of 250 nM means that it is not sensitive enough to detect antigen proteins at physiologically relevant levels (sub-pM). Surface plasmon resonance is a more sensitive technique but requires larger sample volumes, takes longer, requires more expensive instrumentation, and only reduces the detection limit to 5 nM. Surface-enhanced Raman spectroscopy is far more sensitive, enabling detection of spike protein to sub-picomolar concentrations. Control experiments performed using scrambled aptamers and using bovine serum albumin as an analyte show that this apta-sensing approach is both sensitive and selective, with no appreciable response observed for any controls. Overall, these proof-of-principle results demonstrate that SERS-based aptasensors hold great promise for development into rapid, point-of-use antigen detection systems, enabling mass testing without any need for reagents or laboratory expertise and equipment.

Vibrioferrin production by the food spoilage bacterium Pseudomonas fragi.

Pseudomonas fragi is a meat and milk spoilage bacterium with high iron requirements; however, mechanisms of iron acquisition remain largely unknown. The aim of this work was to investigate siderophore production as an iron acquisition system for P. fragi. A vibrioferrin siderophore gene cluster was identified in 13 P. fragi, and experiments were conducted with a representative strain of this group (F1801). Chromeazurol S assays showed that P. fragi F1801 produced siderophores under iron starvation at optimum growth and refrigeration temperature. Conversely, supplementation of low iron media with 50 µM FeCl3 repressed transcription of the vibrioferrin genes and siderophore production. Disruption of the siderophore receptor (pvuA) caused polar effects on downstream vibrioferrin genes, resulting in impaired siderophore production of the ΔpvuA mutant. Growth of this mutant was compared to growth of a control strain (Δlip) with wild-type vibrioferrin genes in low iron media supplemented with iron chelators 2,2΄-bipyridyl or apo-transferrin. While 25 µM 2,2΄-bipyridyl caused impaired growth of ΔpvuA, growth of the mutant was completely inhibited by 2.5 µM apo-transferrin, but could be restored by FeCl3 addition. In summary, this work identifies a vibrioferrin-mediated iron acquisition system of P. fragi, which is required for growth of this bacterium under iron starvation.

Genomic and metabolic characterization of spoilage-associated Pseudomonas species.

Pseudomonas are common spoilage agents of aerobically stored fresh foods. Their ability to cause spoilage is species- and may be strain-specific. To improve our understanding of the meat and milk spoilage agents Pseudomonas fragi and Pseudomonas lundensis, we sequenced the genomes of 12 P. fragi and seven P. lundensis isolates. These genomes provided a dataset for genomic analyses. Key volatile organic compounds (VOCs) produced or metabolised by the isolates were determined during their growth on a beef paste and where possible, metabolic activity was associated with gene repertoire. Genome analyses showed that the isolates included in this work may belong to more than two Pseudomonas species with possible spoilage potential. Pan-genome analyses demonstrated a high degree of diversity among the P. fragi and genetic flexibility and diversity may be traits of both species. Growth of the P. lundensis isolates was characterised by the production of large amounts of 1-undecene, 5-methyl-2-hexanone and methyl-2-butenoic acid. P. fragi isolates produced extensive amounts of methyl and ethyl acetate and the production of methyl esters predominated over ethyl esters. Some of the P. fragi produced extremely low levels of VOCs, highlighting the importance of strain-specific studies in food matrices. Furthermore, although usually not considered to be denitrifiers, all isolates generated molecular nitrogen, indicating that at least some steps of this pathway are intact.

Characterisation of the Brochothrix thermosphacta sortase A enzyme.

Gram-positive bacteria utilise class A sortases to coat the surface of their cells with a diversity of proteins that facilitate interactions with their environment and play fundamental roles in cell physiology and virulence. A putative sortase A gene was identified in the genome of the poorly studied meat spoilage bacterium Brochothrix thermosphacta. To understand how this bacterium mediates interactions with its environment, an N-terminal truncated, His-tagged variant of this protein (His6-BtSrtA) was expressed and purified. Catalytic activity of recombinant His6-BtSrtA was investigated, including sorting motif recognition of target proteins and bioconjugation activity. Further, the B. thermosphacta genome was examined for the presence of sortase A (SrtA) protein substrates. His6-BtSrtA readily formed intermediate complexes with LPXTG-tagged proteins. Although the reaction was inefficient, nucleophilic attack of the resultant thioacyl intermediates by tri-glycine was observed. Genome examination identified 11 potential SrtA substrates, two of which contained protein domains associated with adherence of pathogens to host extracellular matrix proteins and cells, suggesting the B. thermosphacta SrtA may be indirectly involved in its attachment to meat surfaces. Thus, further work in this area could provide crucial insight into molecular mechanisms involved in the colonisation of meat by B. thermosphacta.

Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation.

Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins.

Ribosomal protein S3 interacts with the NF-κB inhibitor IκBα.

Ribosomal protein S3 (RPS3) is part of nuclear, transcriptionally active and cytoplasmic inhibitory complexes containing NF-κB variant p65. We show that in resting HEK293 cells, RPS3 interacts with NF-κB inhibitor IκBα. In contrast, efficient co-precipitation of p65 with RPS3 was only achieved in the presence of ectopic IκBα. In addition, a strong in vitro interaction was observed between RPS3 and IκBα, while binding between RPS3 and p65 was very weak. Furthermore, IκBα facilitated the reconstitution of p65 and RPS3 into one complex in vitro. Our results suggest that IκBα sequesters not only p65 but also RPS3 in the cytoplasm. This would ensure maintenance of an RPS3 pool for the NF-κB pathway as well as equimolar release of RPS3 and p65 upon stimulation.