Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment

Saccharibacteria are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to the Candidate Phyla Radiation. Comparative genomic analyses suggest convergent evolution of key functions enabling the adaptation of environmental Saccharibacteria to mammalian microbiomes. Currently, our understanding of this environment-to-mammal niche transition within Saccharibacteria and their obligate episymbiotic association with host bacteria is limited. Here, we identified a complete arginine deiminase system (ADS), found in further genome streamlined mammal-associated Saccharibacteria but missing in their environmental counterparts, suggesting acquisition during environment-to-mammal niche transition. Using TM7x, the first cultured Saccharibacteria strain from the human oral microbiome and its host bacterium Actinomyces odontolyticus, we experimentally tested the function and impact of the ADS. We demonstrated that by catabolizing arginine and generating adenosine triphosphate, the ADS allows metabolically restrained TM7x to maintain higher viability and infectivity when disassociated from the host bacterium. Furthermore, the ADS protects TM7x and its host bacterium from acid stress, a condition frequently encountered within the human oral cavity due to bacterial metabolism of dietary carbohydrates. Intriguingly, with a restricted host range, TM7x forms obligate associations with Actinomyces spp. lacking the ADS but not those carrying the ADS, suggesting the acquired ADS may also contribute to partner selection for cooperative episymbiosis within a mammalian microbiome. These data present experimental characterization of a mutualistic interaction between TM7x and their host bacteria, and illustrate the benefits of acquiring a novel pathway in the transition of Saccharibacteria to mammalian microbiomes.

Effects of 405 ± 5-nm LED Illumination on Environmental Stress Tolerance of Salmonella Typhimurium in Sliced Beef

Salmonella Typhimurium is a widely distributed foodborne pathogen and is tolerant of various environmental conditions. It can cause intestinal fever, gastroenteritis and bacteremia. The aim of this research was to explore the effect of illumination with 405 nm light-emitting diodes (LEDs) on the resistance of S. Typhimurium to environmental stress. Beef slices contaminated with S. Typhimurium were illuminated by 405 nm LEDs (18.9 ± 1.4 mW/cm2) for 8 h at 4 °C; controls were incubated in darkness at 7 °C. Then, the illuminated or non-illuminated (control) cells were exposed to thermal stress (50, 55, 60 or 65 °C); oxidative stress (0.01% H2O2 [v/v]); acid stress (simulated gastric fluid [SGF] at pH 2 or 3); or bile salts (1%, 2%, or 3% [w/v]). S. Typhimurium treated by 405 nm LED irradiation showed decreased resistance to thermal stress, osmotic pressure, oxidation, SGF and bile salts. The transcription of eight environmental tolerance-related genes were downregulated by the illumination. Our findings suggest the potential of applying 405 nm LED-illumination technology in the control of pathogens in food processing, production and storage, and in decreasing infection and disease related to S. Typhimurium.

Seleksi Mutan Tanaman Lamtoro (Leucaena leucocephala cv. Tarramba) Tahan Kutu Loncat terhadap Lingkungan Kering pada Rumah Kaca

Forage is the main source of feed for ruminants. Forage consists of two types, namely grass and legumes. Leguminosa is a type of forage as a source of protein. One type of legume that is well known by breeders in Indonesia is lamtoro (Leucaena leucocephala cv. Tarramba). This study was aimed to produce candidate mutants of lamtoro drought resistant to acid stress conditions. The experiment in this study used an unbalanced completely randomized design with 6 treatments of 740 different replications based on different sources of lamtoro plant mutants at different levels (P0: 0 n= 89, P1: 100 gy n= 82, P2: 200 gy n= 153, P3:300 gy n=120, P4: 400 gy n= 244, P5: 500 gy n= 52). Variables observed included plant height, number of stalks, leaf loss and stem diameter. The results showed that lamtoro plants irradiated with gamma rays at a level of 200 gy-500 gy were significantly higher than 100 gy irradiation at 10 and 12 days watering, but the diameter of the plants was larger at 100 gy irradiation. It can be watering intervals of 10 days and 12 days on the parameters of height and stem diameter showed that gamma rays irradiation of 400 gy resulted in dry-resistant lamtoro mutant candidates under acid stress condition. Key words: gamma rays, lamtoro mutant, watering interval

Involvement of Small Colony Variant-Related Heme Biosynthesis Genes in Staphylococcus aureus Persister Formation in vitro

Background: Persisters are important reasons for persistent infections, and they can lead to antibiotic treatment failure in patients and consequently chronic infection. Staphylococcus aureus small colony variants (SCVs) have been shown to be related to persistent infection. Mutations in the genes of the heme biosynthesis pathway lead to the formation of SCVs. However, the relationship between heme production genes and persister has not been tested.Methods:HemA and hemB were knocked out by allelic replacement from S. aureus strain USA500 separately, and then, the heme deficiency was complemented by overexpression of related genes and the addition of hemin. The stress-related persister assay was conducted. RNA-sequencing was performed to find genes and pathways involved in heme-related persister formation, and relative genes and operons were further knocked out and overexpressed to confirm their role in each process.Results: We found that heme biosynthesis deficiency can lead to decreased persister. After complementing the corresponding genes or hemin, the persister levels could be restored. RNA-seq on knockout strains showed that various metabolic pathways were influenced, such as energy metabolism, amino acid metabolism, carbohydrate metabolism, and membrane transport. Overexpression of epiF and operon asp23 could restore USA500∆hemA persister formation under acid stress. Knocking out operon arc in USA500∆hemA could further reduce USA500∆hemA persister formation under acid and oxidative stress.Conclusion: Heme synthesis has a role in S. aureus persister formation.

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σB), whose role in protection against acid stress is well established. However, the mechanisms leading to its activation by low pH are unknown. Here, we investigated the involvement of the stressosome, a sensory organelle, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σB and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was present but its remaining paralogues were genetically inactivated retained the ability to induce σB activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1 T175A, T209A, T241A) and RsbS (rsbS S56A), or in the active site of the stressosome kinase RsbT (rsbT N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1 T175A variant, retained a near-wild type phenotype. The rsbR1 T209A and rsbR1 T241A mutants displayed constitutive σB activity. Mild acid shock upregulates invasion genes and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σB in L. monocytogenes. Furthermore, RsbR1 can function independently of its paralogues and that signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.

Investigation of the polyamine biosynthetic and transport capability of Streptococcus agalactiae: the non-essential PotABCD transporter

Polyamines constitute a group of organic polycations positively charged at physiological pH. They are involved in a large variety of biological processes, including the protection against physiological stress. In this study, we show that the genome of Streptococcus agalactiae , a commensal bacterium of the intestine and the vagina and one of the most common agents responsible of neonate infections, does not encode proteins homologous to the specific enzymes involved in the known polyamine synthetic pathways. This lack of biosynthetic capability was verified experimentally by TLC analysis of the intracellular content of S. agalactiae grown in the absence of polyamines. However, similar analyses showed that the polyamines spermidine, spermine and putrescine can be imported from the growth media into the bacteria. We found that all strains of S. agalactiae possess the genes encoding the polyamine ABC transporter PotABCD. We demonstrated that these genes form an operon with folK, a gene involved in folate biosynthesis, murB, a gene involved in peptidoglycan biosynthesis, and with clc, a gene encoding a Cl−/H+ antiporter involved in resistance to acid stress in Escherichia coli . Transcription of the potABCD operon is induced by peroxide-induced oxidative stress but not by acidic stress. Spermidine and spermine were found to be inducers of potABCD transcription at pH 7.4 whereas putrescine induces this expression only during peroxide-induced oxidative stress. Using a deletion mutant of potABCD, we were nevertheless unable to associate phenotypic traits to the PotABCD transporter, probably due to the existence of one or more as yet identified transporters with a redundant action.

Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt’s minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.