NrnA is a 5'-3' exonuclease that processes short RNA substrates in vivo and in vitro.

Bacterial RNases process RNAs until only short oligomers (2-5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2-4 nucleotides in length, all of which were processed from their 5' terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability.

ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.

Choosing the Right Essential Oil for a Mouthwash: Chemical, Antimicrobial and Cytotoxic Studies.

Thirteen commercial essential oils were assessed for their possible inclusion in a mouthwash formulation based on their inhibitory effect against potentially pathogenic anaerobic oral bacterial isolates from subgingival plaque, and their cytotoxicity towards gingival cells. The essential oils, originating from species belonging to seven major aromatic plant families, were chosen to provide the necessary diversity in chemical composition that was analyzed in detail by GC and GC/MS. Multivariate statistical analysis, performed using the in vitro microbiological/toxicological assays and compositional data, revealed that the major components of the essential oils were probably not the main carriers of the activities observed. A formulation of 'designer' mouthwashes is proposed based on the selective action of certain essential oils towards specific bacterial isolates (e. g., Citrus bergamia vs. Parvimonas micra), and non-toxicity to gingival cells at antimicrobially active concentrations.

Clinical characteristics of bloodstream infection by Parvimonas micra: retrospective case series and literature review.

BACKGROUND: Gram-positive anaerobic (GPA) bacteria inhabit different parts of the human body as commensals but can also cause bacteremia. In this retrospective observational study, we analyzed GPA bacteremia pathogens before (2013-2015) and after (2016-2018) the introduction of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). METHOD: We conducted a retrospective observational study by searching the microbiology database to identify all positive GPA blood cultures of patients with GPA bacteremia diagnosed using the new technique, MALDI-TOF MS, between January 1, 2016 and December 31, 2018; and using a conventional phenotypic method between January 1, 2013 and December 31, 2015 at a single tertiary center in Japan. Parvimonas micra (P. micra) (17.5%) was the second most frequently identified GPA (MALDI-TOF MS); we then retrospectively reviewed electronic medical records for 25 P. micra bacteremia cases at our hospital. We also conducted a literature review of published cases in PubMed from January 1, 1980, until December 31, 2019; 27 cases were retrieved. RESULTS: Most cases of P. micra bacteremia were identified after 2015, both, at our institute and from the literature review. They were of mostly elderly patients and had comorbid conditions (malignancies and diabetes). In our cases, laryngeal pharynx (7/25, 28%) and gastrointestinal tract (GIT; 6/25, 24%) were identified as the most likely sources of bacteremia; however, the infection source was not identified in 9 cases (36%). P. micra bacteremia were frequently associated with spondylodiscitis (29.6%), oropharyngeal infection (25.9%), intra-abdominal abscess (14.8%), infective endocarditis (11.1%), septic pulmonary emboli (11.1%), and GIT infection (11.1%) in the literature review. Almost all cases were treated successfully with antibiotics and by abscess drainage. The 30-day mortalities were 4 and 3.7% for our cases and the literature cases, respectively. CONCLUSIONS: Infection sites of P. micra are predominantly associated with GIT, oropharyngeal, vertebral spine, intra-abdominal region, pulmonary, and heart valves. Patients with P. micra bacteremia could have good prognosis following appropriate treatment.

The czcD (NiCo) Riboswitch Responds to Iron(II).

Iron is essential for nearly every organism, and mismanagement of its intracellular concentrations (either deficiency or excess) contributes to diminished virulence in human pathogens, necessitating intricate metalloregulatory mechanisms. To date, although several metal-responsive riboswitches have been identified in bacteria, none has been shown to respond to FeII. The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to NiII and CoII but thought not to respond to FeII, on the basis of aerobic, in vitro assays; its function in vivo is not well understood. We constructed a fluorescent sensor using this riboswitch fused to the RNA aptamer, Spinach2. When assayed anaerobically, the resulting sensor responds in vitro to FeII, as well as to MnII, CoII, NiII, and ZnII, but only in the cases of FeII and MnII do the apparent Kd values (0.4 and 11 µM, respectively) fall within the range of labile metal concentrations maintained by known metalloregulators. We also show that the sensor-which is, to the best of our knowledge, the first reversible genetically encoded fluorescent sensor for FeII-responds to iron in Escherichia coli cells. Finally, we demonstrate that the putative metal exporters directly downstream of two czcD riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results indicate that iron merits consideration as a plausible physiological ligand for czcD riboswitches, although a response to general metal stress cannot be ruled out at present.

Construction of a Macrophage-Targeting Bio-nanocapsule-Based Nanocarrier.

The construction protocol of bio-nanocapsule (BNC)-based nanocarriers, named GL-BNC and GL-virosome, for targeted drug delivery to macrophages is described here. First, genes encoding the Streptococcus sp. protein G-derived C2 domain (binds to IgG Fc) and Finegoldia magna protein L-derived B1 domain (binds to Igκ light chain) are prepared by PCR amplification. Subsequently, the genes encoding hepatic cell-specific binding domain of hepatitis B virus envelope L protein are replaced by these PCR products. The expression plasmid for this fused gene (encoding GL-fused L protein) can be used to transform Saccharomyces cerevisiae AH22R- cells. To obtain GL-BNC, the transformed yeast cells are disrupted with glass beads, treated with heat, and then subjected to IgG affinity column chromatography followed by size exclusion column chromatography. In addition, GL-BNCs can be fused with liposomes to form GL-virosome. The targeted delivery of GL-BNC and GL-virosome to macrophages can be confirmed by in vitro phagocytosis assays using the murine macrophage cell line RAW264.7.

Phylogenetic Analysis and Screening of Antimicrobial and Antiproliferative Activities of Culturable Bacteria Associated with the Ascidian Styela clava from the Yellow Sea, China.

Over 1,000 compounds, including ecteinascidin-743 and didemnin B, have been isolated from ascidians, with most having bioactive properties such as antimicrobial, antitumor, and enzyme-inhibiting activities. In recent years, direct and indirect evidence has shown that some bioactive compounds isolated from ascidians are not produced by ascidians themselves but by their symbiotic microorganisms. Isolated culturable bacteria associated with ascidians and investigating their potential bioactivity are an important approach for discovering novel compounds. In this study, a total of 269 bacteria were isolated from the ascidian Styela clava collected from the coast of Weihai in the north of the Yellow Sea, China. Phylogenetic relationships among 183 isolates were determined using their 16S rRNA gene sequences. Isolates were tested for antimicrobial activity against seven indicator strains, and an antiproliferative activity assay was performed to test for inhibition of human hepatocellular carcinoma Bel 7402 and human cervical carcinoma HeLa cell proliferation. Our results showed that the isolates belonged to 26 genera from 18 families in four phyla (Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes). Bacillus and Streptomyces were the most dominant genera; 146 strains had potent antimicrobial activities and inhibited at least one of the indicator strains. Crude extracts from 29 strains showed antiproliferative activity against Bel 7402 cells with IC50 values below 500 µg·mL-1, and 53 strains showed antiproliferative activity against HeLa cells, with IC50 values less than 500 µg·mL-1. Our results suggest that culturable bacteria associated with the ascidian Styela clava may be a promising source of novel bioactive compounds.

Polymicrobial anaerobic bacteremia due to Atopobium rimae and Parvimonas micra in a patient with cancer.

Atopobium rimae and Parvimonas micra are both Gram-positive anaerobes involved infrequently in human infections. We report a polymicrobial anaerobic bacteremia caused by these microorganisms. A 43-year-old woman receiving coadjuvant chemotherapy due to a retroperitoneal leiomiosarcoma presented with nausea, vomiting, abdominal pain and fever (38 °C). The two blood cultures resulted in isolation of A. rimae and P. micra, being identified at species level by matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) technology with high log scores. The microorganisms were susceptible to penicilllin, amoxicillin-clavulanate, piperacillin-tazobactam, clindamycin, metronidazole, imipenem, and moxifloxacin. Treatment with levofloxacin was started and subsequently it was changed to piperacillin/tazobactam plus metronidazole and completed for 10 days, but the patient died days later due to her underlying disease.