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Thermophilic members of the genus Aneurinibacillus constitute a remarkable group of microorganisms that exhibit extraordinary flexibility in terms of polyhydroxyalkanoates (PHA) synthesis. In this study, we demonstrate that these Gram-positive bacteria are capable of the utilization of selected lactones, namely, γ-valerolactone (GVL), γ-hexalactone (GHL), and δ-valerolactone (DVL) as the structural precursors of related PHA monomers. In the presence of GVL, a PHA copolymer consisting of 3-hydroxybutyrate, 3-hydroxyvalerate, and also 4-hydroxyvalerate was synthesized, with a 4 HV fraction as high as 53.1 mol%. Similarly, the application of GHL resulted in the synthesis of PHA copolymer containing 4-hydroxyhexanaote (4HHx) (4HHx fraction reached up to 11.5 mol%) and DVL was incorporated into PHA in form of 5-hydroxyvalerate (5 HV) (maximal 5 HV content was 44.2 mol%). The produced materials were characterized by thermoanalytical and spectroscopic methods; the results confirmed extremely appealing material properties of produced copolymers. Further, due to their unique metabolic features and capability of incorporating various PHA monomers into the PHA chain, thermophilic Aneurinibacillus spp. can be considered not only promising chassis for PHA production but also potential donors of PHA-relevant genes to improve PHA production in other thermophiles by using approaches of synthetic biology.

The microorganisms living on the phyllosphere (the aerial part of the plants) are in contact with the lignocellulosic plant cell wall and might have a lignocellulolytic potential. We isolated a Saccharibacillus strain (Saccharibacillus WB17) from wheat bran phyllosphere and its cellulolytic and hemicellulolytic potential was investigated during growth onto wheat bran. Five other type strains from that genus selected from databases were also cultivated onto wheat bran and glucose. Studying the chemical composition of wheat bran residues by FTIR after growth of the six strains showed an important attack of the stretching C-O vibrations assigned to polysaccharides for all the strains, whereas the C = O bond/esterified carboxyl groups were not impacted. The genomic content of the strains showed that they harbored several CAZymes (comprised between 196 and 276) and possessed four of the fifth modules reflecting the presence of a high diversity of enzymes families. Xylanase and amylase activities were the most active enzymes with values reaching more than 4746 ± 1400 mIU/mg protein for the xylanase activity in case of Saccharibacillus deserti KCTC 33693

Several members of Cohnella genus have been reported as xylanolytic bacteria with significant capacity as carbohydrate-active enzyme producers (CAZymes), whose mechanisms involving xylan degradation are a key goal for suitable applications in bio-based industries. Using Cohnella sp. AR92 bacterium, we ensembled a genomic-proteomic approach to assess plant biomass conversion targeting its xylanolytic set of enzymes. Also, the genomic traits of the strain AR92 were compared to other Cohnella spp., showing a significant variability in terms of genome sizes and content of genes that code CAZymes. The AR92 strain genome harbours 209 CAZymes encoding sequences active on different polysaccharides, particularly directed towards xylans. Concurrent proteomic data recovered from cultures containing three kinds of lignocellulosic-derived substrates showed a broad set of xylan-degrading enzymes. The most abundant CAZymes expressed in the different conditions assayed were endo-β-1,4-xylanases belonging to the GH11 and GH10 families, enzymes that were previously proved to be useful in the biotransformation of lignocellulosic biomass derived from sugarcane as well as onto xylan-enriched substrates. Therefore, considering the large reserve of CAZymes of Cohnella sp. AR92, a xylan processing model for AR92 strain is proposed.

Bacillus siamensis LDR was tested for its potential as a biocontrol agent against the phytopathogenic fungi Curvularia lunata BM and Ganoderma sp. TB4. Fermentation of B. siamensis LDR for the production of antifungal compound was performed in modified Czapex-Dox broth using hanjeli (Coix lacryma-jobi L.) starch as carbon source. The Bacillus siamensis LDR inoculum was 105 CFU/ mL, and fermentation was conducted for up to 16 days. Antibiosis assay conducted to test the antifungal activity of filtrate medium. The results showed inhibition of C. lunata BM and Ganoderma sp. TB4 were 47.08% and 85.99%, respectively on 14th day of fermentation. Antifungal assay of the crude extract from filtrate medium revealed growth inhibition of C. lunata BM (60.70%) and Ganoderma sp. TB4 (65.25%). Thin layer chromatography of the crude extract revealed pink-colored spots indicative of lipopeptide compounds. Analysis of the crude extract by ultraperformance liquid chromatography-mass spectrometry was tentatively identified as iturin A, bacillomycin F, and surfactin.

Streptococcus mutans (S. mutans) and Staphylococcus aureus (S. aureus) pathogenicity that alter biofim, has become one of risk factor in orthodontic treatment. The medicinal plant’s Butterfly pea efficacy as an antibacterial agent should be confirmed in dentistry. The purpose of this study is to investigate the antibacterial activity of Medan butterfly pea corolla extract (BPCE) against S. mutans ATCC®25175™ and S. aureus ATCC®6538™. This is a laboratory experiment with Post Test Only Group Design. The minimum inhibitory concentration of BPCE is 6.25 mg/mL. The best concentration of butterfly pea extract to inhibit biofilm formation (antibiofilm) is 100 mg/mL. There was a significant difference (p < 0.05) for antibiofilm activity assays and determination of intramembrane cellular leakage. Although Medan BPCE was inadequate enough in forming antibiofilm and caused intramembrane leakage of S. mutans and S. aureus, further studies in exploring the potential morphological traits of these herbs related to orthodontic products are quite promising.

The efficiency of four species of wax degrading bacteria, Serratia marcescens (Bizio) (Enterobacterales:Yersiniaceae), Pseudomonas putida (Trevisan) (Pseudomonadales: Pseudomonadaceae), Bacillus subtilis (Ehrenberg) (Bacillales:Bacillaceae) and Bacillus cereus (Frankland & Frankland) (Bacillales:Bacillaceae) for the management of a major citrus pest, the mealybug, Planococcus citri (Green) (Hemiptera:Pseudococcidae) was evaluated. The degrading ability of the bacteria, isolated from cadavers of P. citri was assessed in vitro on wax extracted from the mealybug, paraffin wax and low-density polyfilm along with lipase activity. Except B. cereus, all bacteria showed significant lethal and debilitating effects against P. citri in laboratory bioassays. Even though, we did not observe any lethal effect on 1st and 2nd instar nymphs, in the latter instars mortality rate could reach as high as 70.0% in case of S. marcescens. Adults that survived bacterial treatments had significantly reduced female longevity, fecundity and adult formation. In greenhouse trail conducted under controlled conditions, it was found that S. marcescens was the most promising bacteria in managing P. citri population after 14 days of application causing around 36.5% mortality of crawlers coupled with a significant reduction in the wax content and weight of mealybug. However, its application followed by a single spray of half a dose of chlorpyrifos at weekly intervals further enhanced the mortality rate up to 54.0%. Therefore, we conclude that sequential application of S. marcescens followed by a single spray of a half a dose of insecticide is effective for management of the P. citri.

AIMS Examine the effect of soy protein concentrate (SPC) on allochthonous microbiota, hindgut integrity, and liver tissue of totoaba (Totoaba macdonaldi). METHODS AND RESULTS Four diets were prepared: control diet (100% fishmeal) and experimental diets containing partial substitution of fishmeal by SPC (15%, 30% and 45% SPC). After 90 days, samples of the hindgut contents were taken to determine the taxonomic composition of the allochthonous microbiota through sequencing of the V3-V4 region of the 16S rRNA gene. Simultaneously, liver and hindgut samples were collected for examination by histological approaches. The SPC modulated the richness and abundance of the accessory microbiota, of which the main operational taxonomic unit showed an increase corresponding to the Phylum Firmicutes (Bacillales and Lactobacillales). With the increase in SPC, a slight decrease in mucosal fold width, a decrease in goblet cells and a slight distortion of the villi in the hindgut were observed. In the liver, SPC was observed to influence hepatocytes morphology through irregular and enlarged nuclei. CONCLUSION The study demonstrates that Proteobacteria dominated the allochthonous microbiota of subadult totoaba, regardless of the diet. However, the SPC modulated the accessory bacteria communities and caused slight effects on the liver and gut of fish. SIGNIFICANCES AND IMPACT OF THE STUDY To our knowledge, this is the first study that analyses the effects of SPC on allochthonous microbiota of subadults T. macdonaldi through new generation techniques such as DNA sequencing for metagenomic analysis.

SR1 is a dual-function sRNA from Bacillus subtilis. It inhibits translation initiation of ahrC mRNA encoding the transcription activator of the arginine catabolic operons. Base-pairing is promoted by the RNA chaperone CsrA, which induces a slight structural change in the ahrC mRNA to facilitate SR1 binding. Additionally, SR1 encodes the small protein SR1P that interacts with glyceraldehyde-3P dehydrogenase A to promote binding to RNase J1 and enhancing J1 activity. Here, we describe a new target of SR1, kinA mRNA encoding the major histidine kinase of the sporulation phosphorelay. SR1 and kinA mRNA share 7 complementary regions. Base-pairing between SR1 and kinA mRNA decreases kinA translation without affecting kinA mRNA stability and represses transcription of the KinA/Spo0A downstream targets spoIIE, spoIIGA and cotA. The initial interaction between SR1 and kinA mRNA occurs 10 nt downstream of the kinA start codon and is decisive for inhibition. The sr1 encoded peptide SR1P is dispensable for kinA regulation. Deletion of sr1 accelerates sporulation resulting in low quality spores with reduced stress resistance and altered coat protein composition which can be compensated by sr1 overexpression. Neither CsrA nor Hfq influence sporulation or spore properties.

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH⁎, ΔS⁎, and ΔG⁎ were 187 min at 60 °C, 7.3 kJ.mol−1, −76.8 kJ.mol−1.°K−1, and 84.1 kJ.mol−1, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0–10.0) and temperatures (35–75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA–DNA hybridization, digital DNA–DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.

Allsopp, Peter G. (2022): Australian Melolonthini (Coleoptera: Scarabaeidae: Melolonthinae): reclassification of eight species to Antitrogus Burmeister, 1855 and Barryfilius new genus. Zootaxa 5213 (5): 513-545, DOI: https://doi.org/10.11646/zootaxa.5213.5.3

Barryfilius vernus (Britton, 1978), new combination (Figs. 18, 24, 25) Lepidiota verna Britton, 1978: 66, figs. 201–202 (aedeagus), fig. 282D (head and pronotum). Type series. Holotype male in ANIC: Paluma (Mt Spec), 6.i.1969 J.G. Brooks [18.98°S, 146.22°E] (examined). Paratypes: 3 mi. [5 km] SSW of Millaa Millaa [17.56°S, 145.60°E], 22.viii.1967, R.J. Elder (ANIC); 10&male;, Malaan [17.61°S, 145.59°E], 30.viii.1965, 14.ix.1965, 21.ix.1965, 28.ix.1965, 10.x.1965, 18.x.1965, 19.x.1965, 4.xi.1965, 13.ix.1966, 11.x.1966, 18.x.1966, R.J. Elder (QDAF); 9&male;, Malaan, 8 mi. [12.9 km] E of Ravenshoe, 23.vii.1968, 6.viii.1968, 13.viii1968, 17.ix.1968, 1.x.1968, 15.x.1968, R.J. Elder (ANIC); 1&male;, Paluma Dam [18.96°S, 146.15°E], 24.xii.1963, G. Monteith (UQIC); 1&male;, Birthday Ck Falls, 6.7 m [10.7 km] NW Paluma [18.98°S, 146.16°E], 11.xii.1972, J.G. Brooks (ANIC); 1&male;, Mt Spec Nat. Pk. via Paluma [18.98°S, 146.22°E], 7.xi.1969, T. Weir (UQIC); 1&male;, Mt Spec, 9.i.1968, J.G. Brooks (ANIC); 6&male;, Paluma (Mt Spec), 6.i.1969, 7.i.1969, J.G. Brooks (ANIC); 1&male;, Star Valley Lookout 2.3 m [3.7 km] W. Paluma [19.01°S, 146.18°E], 12.xii.1972, J.G. Brooks (ANIC). Other material examined. AUSTRALIA: Queensland: 1&male;, Massey Creek BS3 [17.37°S, 145.34°E], 3.x- 2.xi.1995, L. Umback (ANIC); 3&male;, Millaa Millaa [17.51°S, 145.61°E], 7.ix.1965, R.J. Elder (QDAF); 1&male;, 1&male;, 3 mi. [5 km] SSW of Millaa Millaa, 17.ix.1968, R.J. Elder (ANIC); 1&female;, South Johnstone, 20.xi.1966, G. Wilson (ANIC, bred out); 1&male;, Mt Haig GS1 [17.96°S, 145.36°E], 31.vii-31.viii.1995, L. Umback (ANIC); 2&male;, Mt Spec S3 [18.55°S, 146.09°E], 6.ii.-9.iii.1995, M. Cermak (ANIC); 1&male;, Mt Spec S2 [18.55°S, 146.10°E], 10.i-6.ii.1995, M. Cermak (ANIC); 9&male;, Paluma [19.00°S 146.12°E], xi-xii.1978, 22.x.1979, xii1980 - i.1981, D.W. Frith (ANIC); 1&male;, Paluma, 17-18.xii.1988, H. & A. Howden (CMN); 2&male;, Paluma, 12.xii.1972, J.G. Brooks (ANIC); 13&male;, Mt. Spec S1 [19.00°S, 146.11°E], 6.xii.1994 - 10.i.1995, M. Cermak (ANIC). Remarks. One of the paratypes in UQIC has Britton’s 1969 paratype label but with the unpublished name ‘ Lepidiota palumae’—he evidently decided later to change the name. Of the paratypes collected by Brooks at Paluma, Britton (1978) listed only the date 6.i.1969; the one collected a day later is labelled as a paratype. Britton (1978) gave no etymology for the specific name. There are two possibilities: a noun, verna (slave born in the house), that should retain its nominative form and not be changed, and that would make sense if the beetle inhabits people’s houses; or, an adjective that relates to the season Spring. Given that some specimens were collected in October, the second seems more likely, so I have assumed the adjectival form with the appropriate change in ending. Diagnosis. Male. Body 20–22 mm long; very dark red-brown (Fig. 18). Terminal palpomere of maxillary palp large, 1.1 mm long, elongate-elliptical (length:width 3.3:1), with large, flat, longitudinally strigose, elliptical depression on dorsal side. Clypeus with anterior face moderately deep, width 5x length; upper surface transverse, width 3.1–3.2x length, anterior margin slightly indented in middle, with broadly rounded angles; upper surface strongly concave, coarsely and densely punctured, each of the broad, flat-bottomed punctures with flattened, white seta about as long as radius of the puncture. Frons irregularly and coarsely punctured anteriorly of a slight ridge, each puncture with a short, white seta as long as the radius of the puncture; posterior quarter with only a few punctures and setae; without setae above each eye. Antennae with 10 antennomeres, antennomere 3 elongate-cylindrical, antennomere 4 discoidal, antennomere 5 discoidal but with slight swelling on inner edge, antennomeres 6–7 with a very short lamella, antennomeres 8–10 lamellate, 2.7–2.8 mm long. Pronotum transverse, greatest width 1.6x length; anterior margin narrow, continuous and raised, punctate except in middle; lateral edges with rounded, obtuse angle slightly behind middle, straight before angle and slightly concave after; posterior margin with narrow, raised margin but absent in middle and near posterior angles; anterior angles square, posterior angles slightly obtuse; surface uniformly punctured (approximately 18 mm-2), each puncture with minute, slightly elongate, white seta about as long as diameter of the puncture, punctures and setae denser along lateral margins behind angle, surface smooth and shiny between punctures. Scutellum with line of dense, setose punctures close to lateral edge, disc sparsely punctured with setae as on pronotum, margins slightly raised and defined by a groove. Elytra sparsely but fairly uniformly punctured (approximately 10 mm-2) except on sutural interval, each puncture with sort, ovoid, white seta (0.06 mm long), as long as those on pronotum. Propygidium with few punctures with short setae across posterior and lateral margins. Pygidium irregularly punctured, approximately 25 mm- 2 in middle, with setae similar to elytra, about 2.5–3.0x long as wide, surface between punctures microreticulate, long, yellow setae on posterior-lateral margins. Ventral surface of thorax densely clothed with long, fine, yellow setae; anterior half of pronotal hypomeron with elongate, white setae near lateral margins, remainder of anterior half with longer, yellow-white setae. Teeth on outer edge of protibia medium-short and rounded. Ventrites sparsely clothed with short, setae similar to elytra; first visible ventrite with longer, yellow setae anteriorly and laterally; penultimate ventrite with a few, scattered, long, yellow setae on posterior half; terminal ventrite without lateral lobes, with long setae along posterior margin. Aedeagus virtually symmetrical, in lateral view each paramere constricted at about half length then widening to apex (Fig. 24). Female. Unknown. Distribution (Fig. 25). Occurs from the southern part of the Atherton tableland south to Paluma. The female bred out by George Wilson and labelled ‘South Johnstone’ most likely originated from the uplands of the East Palmerston or Nerada area (about 17.58°S, 145.85°E) and not the lowland village of South Johnstone where Wilson had his office. Natural history. Adult males have been collected at light and in oil-bath and flight-intercept traps mainly during November-January, but with a set of records from Maalan for January and August–October (Britton 1978)., Published as part of Allsopp, Peter G., 2022, Australian Melolonthini (Coleoptera: Scarabaeidae: Melolonthinae): reclassification of eight species to Antitrogus Burmeister, 1855 and Barryfilius new genus, pp. 513-545 in Zootaxa 5213 (5) on pages 528-530, DOI: 10.11646/zootaxa.5213.5.3, http://zenodo.org/record/7381670, {"references":["Britton, E. B. (1978) A revision of the Australian chafers (Coleoptera: Scarabaeidae: Melolonthinae). Vol. 2. Tribe Melolonthini. Australian Journal of Zoology, Supplementary Series, 60, 1 - 150. https: // doi. org / 10.1071 / AJZS 060"]}

Barryfiliusa quinarius (Britton, 1978), new combination (Figs. 17, 23, 25) Lepidiota quinaria Britton, 1978: 64, figs. 193–194 (aedeagus), fig. 281F (head and pronotum); Weir et al. 2019: fig. 173 (head). Type series. Holotype male in ANIC: Koombooloombah [Koombooloomba] [17.83°S, 145.60°E], Queensland, i.54, J.G. Brooks (examined). Paratypes: 2&male;, 14 m [miles, 22.5 km] up Whitfield Range Rd, Cairns, 5.i.1972, A Walford-Huggins (ANIC); 1&male;, Ringrose Nat. Pk. via Atherton [Mount Hypipamee National Park, 17.43°S, 145.49°E], 9.xii.1966, B. Cantrell (UQIC); 1&male;, Ravenshoe [17.61°S, 145.49°E], 11.i.1962, E.B. Britton (ANIC); 1&male;, Koombooloomba, i.1954, GB (ANIC); 1&male;, Koombooloomba, 5.i.1954, J.G. Brooks (ANIC). Other material examined. AUSTRALIA: Queensland: 1&male;, Mt Lewis [16.58°S, 145.28°E], 29.xii.1979, P. Carne (ANIC); 1&male;, 8 miles [12.9 km] up Whitfield Range Rd, Cairns [16.95°S, 145.71°E], Walford-Huggins (QDAF); 2 &male;, 12 miles [19.3 km] Whitfield Range Rd, Cairns, 5.i.1972, A. & M. Walford-Huggins (CMN); 3&male;, Davies Creek, 20 km SE of Mareeba [16.98°S, 145.55°E], 25.xii.1988, H. & A. Howden (CMN); 1&male;, Mt Bartle Frere [17.38°S, 145.82°E], 7.x.1969, A. & M. Walford-Huggins (CMN); 1&male;, Cardwell Range, Upper Broadwater Creek Valley [18.50°S, 146.00°E], 17–21.xii.1984, Monteith, Thompson & Hamlet (QM); 4&male;, Kirrama Range, Douglas Creek Rd [18.70°S, 145.72°E], 9–12.xii.1986, Monteith, Thompson & Hamlet (QM). Comment. The paratype from Ravenshoe was listed by Britton (1978) as in NHML. Diagnosis. Male. Body 23–26 mm long; uniformly dark red-brown (Fig. 17). Terminal palpomere of maxillary palp large, 1.15 mm long, elongate-elliptical (length:width 3.5:1), with large, flat, dull, elliptical depression on dorsal side. Clypeus (Weir et al. 2019: fig. 173) with anterior face moderately deep, width 6.7x length; upper surface strongly transverse, width 3.7–4.0x length, anterior margin slightly indented in middle, with rounded angles; upper surface strongly concave, densely punctured, each of the broad, flat-bottomed punctures with a flattened, white seta about as long as radius of the puncture. Frons irregularly and coarsely punctured, each puncture with a short, white seta as long as the radius of the puncture; posterior quarter with only a few punctures and setae; antero-laterally with a few, recumbent, yellow setae above each eye. Antennae with 10 antennomeres, antennomere 3 elongate-cylindrical, antennomere 4 discoidal, antennomere 5 lamellate but lamella 0.33–0.5x long as that of antennomere 6, antennomere 6–10 lamellate, 2.8 mm long. Pronotum transverse, greatest width 1.50–1.65x length; anterior margin narrow, continuous and raised, punctate except in middle; lateral edges with a rounded, obtuse angle in middle, straight before angle and slightly concave after; posterior margin with a narrow, raised margin in middle; anterior and posterior angles slightly obtuse; surface uniformly punctured (approximately 17 mm-2), each puncture with minute, slightly elongate, white seta about as long as diameter of the puncture, punctures and setae denser along posterior margin and lateral margins behind angle. Scutellum with line of small punctures close to lateral edge, disc sparsely punctured with setae as on pronotum. Elytra sparsely but fairly uniformly punctured (approximately 10 mm-2) except on sutural interval, each puncture with stout, acutely pointed, white seta (0.06 mm long), as long as those on pronotum. Propygidium with a few punctures with short setae across posterior and lateral margins. Pygidium irregularly punctured, approximately 30 mm- 2 in middle; setae short but more elongate than on elytra, surface between punctures microreticulate, long, yellow setae on posterior-lateral margins. Ventral surface of thorax densely clothed with long, fine, yellow setae; pronotal hypemeron also with elongate, white setae near lateral margins. Teeth on outer edge of protibia long and acute. Ventrites sparsely clothed with short, setae similar to elytra; first visible ventrite with longer, yellow setae; penultimate ventrite with a few, scattered, long, yellow setae near posterior margin; terminal ventrite without lateral lobes. Aedeagus slightly asymmetrical, mid inner margin of one paramere crossing over the other (Fig. 23). Female. Unknown. Distribution (Fig. 25). Known from northeastern Queensland from Mount Lewis, south through the eastern and southern edges of the Atherton Tableland to the northern side of the Herbert River Valley. This species crosses the Black Mountain Corridor (Bryant & Krosch 2016), a gap of 60 km between areas of higher elevation, that today retains pockets of rainforest interspersed among drier habitats, and indicates broader elevational and/or habitat tolerances different from those of B. contiguus, which is restricted to only one of the northeastern highland areas. Similar patterns occur in other groups of animals, including some Scarabaeidae (Reid 2000; Reid & Storey 2000). Natural history. Adult males were mainly collected in December–January at light (Britton 1978), with one record from October. One was collected on eucalypt flowers., Published as part of Allsopp, Peter G., 2022, Australian Melolonthini (Coleoptera: Scarabaeidae: Melolonthinae): reclassification of eight species to Antitrogus Burmeister, 1855 and Barryfilius new genus, pp. 513-545 in Zootaxa 5213 (5) on pages 527-528, DOI: 10.11646/zootaxa.5213.5.3, http://zenodo.org/record/7381670, {"references":["Britton, E. B. (1978) A revision of the Australian chafers (Coleoptera: Scarabaeidae: Melolonthinae). Vol. 2. Tribe Melolonthini. Australian Journal of Zoology, Supplementary Series, 60, 1 - 150. https: // doi. org / 10.1071 / AJZS 060","Weir, T. A., Lawrence, J. F., Lemann, C. & Gunter, N. L. (2019) Scarabaeidae: Melolonthinae Leach, 1819. In: Slipinski, A. & Lawrence, J. F. (Eds.), Australian Beetles. Vol. 2. Archostemata, Myxophaga, Adephaga, Polyphaga (part). CSIRO Publishing, Clayton South, pp. 467 - 507.","Bryant, L. M. & Krosch, M. N. (2016) Lines in the land: a review of evidence for eastern Australia's major biogeographical barriers to closed forest taxa. Biological Journal of the Linnean Society, 119, 238 - 264. https: // doi. org / 10.1111 / bij. 12821","Reid, C. A. M. & Storey, R. I. (2000) Revision of the dung beetle genus Temnoplectron Westwood (Coleoptera: Scarabaeidae: Scarabaeini). Memoirs of the Queensland Museum, 46, 253 - 297."]}

Barryfilius lewisae (Britton, 1978), new combination (Figs. 16, 22, 25) Lepidiota lewisae Britton, 1978: 67, figs. 203–205 (aedeagus), fig. 282E (head and pronotum). Type series. Holotype male in QM: Mount Lewis, via Julatten, N. Qld. [16.57°S, 145.28°E], 3,500-4000’ [feet], 27–28.xi.1965, G. Monteith (examined). Diagnosis. Male. Body 22 mm long; dark red-brown, pronotum darker than elytra (Fig. 16). Terminal palpomere of maxillary palp large, 1 mm long, elongate-elliptical (length:width 3.3:1), with a large, flat, dull, elliptical depression on dorsal side. Clypeus with anterior face moderately deep, width 5x length; upper surface transverse, width 2.7x length, anterior margin slightly indented in middle, with rounded angles; upper surface strongly concave, densely punctured, each of the broad, flat-bottomed punctures with a flattened, white seta about as long as radius of the puncture. Frons irregularly and coarsely punctured, each puncture with a short, white seta as long as the radius of the puncture; posterior quarter with only a few punctures and setae; antero-laterally without longer setae above each eye. Antennae with 10 antennomeres, antennomere 3 elongate-cylindrical, antennomere 4 discoidal, antennomeres 5–7 each with a very short indication of a lamella, antennomeres 8–10 lamellate, 2.8–2.9 mm long. Pronotum transverse, greatest width 16x length; anterior margin narrow, continuous and raised, punctate except in middle; lateral edges with a rounded, obtuse angle in middle, straight before and after angle; posterior margin with a narrow, raised margin, slightly obliterated in the middle and towards posterior angle; anterior and posterior angles slightly obtuse; surface uniformly punctured (approximately 6–8 mm-2), denser towards anterior edge, especially in middle and in angles, each puncture with a minute slightly elongate, white seta about as long as diameter of the puncture. Scutellum with a line of small punctures close to lateral edge in anterior third, disc sparsely punctured with setae as on pronotum. Elytra sparsely but fairly uniformly punctured (approximately 10 mm-2) except on sutural interval, each puncture with a stout, acutely pointed, white seta as long as those on pronotum. Propygidium with a few scattered punctures with short setae. Pygidium similarly punctured; setae short but more elongate than on elytra, surface between punctures microreticulate, long, yellow setae on posterior-lateral margins. Ventral surface of thorax densely clothed with long, fine, yellow setae; pronotal hypemeron also with elongate, white setae. Teeth on outer edge of protibia rounded. Ventrites clothed with short, setae similar to pygidium, sparser on posterior ventrites; first visible ventrite with longer, yellow setae; penultimate ventrite with a few, scattered, long, yellow setae near posterior margin; terminal ventrite without lateral lobes. Aedeagus slightly asymmetrical, apices rounded in dorsal view (Fig. 22). Female. Unknown. Distribution (Fig. 25). Known only from Mount Lewis northwest of Julatten, an isolated massive south of the Windsor Tableland where B. contigua occurs. Natural history. The type was taken in late November (Britton 1978)., Published as part of Allsopp, Peter G., 2022, Australian Melolonthini (Coleoptera: Scarabaeidae: Melolonthinae): reclassification of eight species to Antitrogus Burmeister, 1855 and Barryfilius new genus, pp. 513-545 in Zootaxa 5213 (5) on page 527, DOI: 10.11646/zootaxa.5213.5.3, http://zenodo.org/record/7381670, {"references":["Britton, E. B. (1978) A revision of the Australian chafers (Coleoptera: Scarabaeidae: Melolonthinae). Vol. 2. Tribe Melolonthini. Australian Journal of Zoology, Supplementary Series, 60, 1 - 150. https: // doi. org / 10.1071 / AJZS 060"]}

Barryfilius laevis (Arrow, 1932), new combination (Figs. 15, 21, 25) Lepidiota laevis Arrow, 1932: 194; Britton 1978: 66, figs. 199–200 (aedeagus), fig. 282C (head and pronotum). Type series. Lectotype male in NHML (designated by Allsopp 2020b: 465): Type (circular red-ringed, typeset) | Pest in dairying and maize-growing district (handwritten) | AUSTRALIA N. Queensland J.N. Smith (typeset) | &male; (typeset) | Loc. Atherton [17.27°S, 145.58°E] 2.1.30 S (handwritten) / Pres. By Imp. Inst. Ent. Brit. Mus. 1931-469 (typeset) | Lepidiota laevis type Arrow (handwritten) | my 2019 lectotype label. Paralectotypes: AUSTRALIA: Queensland: 1&male;, Atherton, 2.i.1930, J.N. Smith (QM); 1&male;, Atherton 7.iii.1931, J.N. Smith (NHML); 1&female;, N. Queensland, Mareeba S, 3.i.1934, J.N. Smith (ANIC). Other material examined. AUSTRALIA: Queensland: 4&male;, Atherton, 4.i.1898, 2.i.1930, 2.i.1931, 22.i.1931 (ANIC, QDAF); 1&male;, Atherton, 2.i.1930, QDAS [Queensland Department of Agriculture and Stock] (ANIC); 1&male;, Atherton, 3.i.1931, J.H. Smith (QDAF); 1&male;, Atherton, 11.ii.1959, GWS [G.W. Saunders] (QDAF); 1&male;, Atherton, 14.xii.1958 G.E [G. Ettershank] (QM); 10&male;, Atherton, 24.ix.1958, 25.xii.1959, 27.xii.1959, G. Ettershank (QDAF, QM); 1&male;, Atherton, i.1960, CV (ANIC); 6&male;, Atherton, 9.i.1968, P. Ferrar (ANIC); 5&male;, Atherton, 3.i.1969, 16.i.1969, 27.i.1969, 29.i.1969, J. Barrett (CMN, QDAF); 1&male;, Atherton, 16.xii.1969, G. Saunders (QDAF); 1&male;, Atherton, ii.1976, W. Crees (QM);. 1&male;, Atherton, 15.xii.1976, A.K. Irvine (ANIC); 11&male;, Newell St, Atherton [17.27°S 145.47°E], 23.xii.1969, 24.xii.1969, 30.xii.1969, 20.i.1970, 22.i.1970, 23.xii.1970, 28.xii.1973, JHB or J.H. Barrett (CMN, QDAF); 2&male;, 2 km NE Atherton, 20.xii.1997, D.J.W. Burgess, at light (QM); 23&male;, 8&female;, 2.5 m [4 km] S. Atherton [17.29S, 145.47E], 12.v.1970 [larvae collected], 19.i.1971 [adults emerged], R.J. Roberts (ANIC); 1&male;, Kureen [17.34°S, 145.59°E], 4.i.1931 (QDAF); 1&male;, 179 Sewell Rd, Lake Eacham [17.29°S, 145.66°E], 18- 25.ii.2020, K.J. Chandler, to light (QM); 1&male;, Millaa Milla [17.51 145.61E], 7.ix.1965, RJE [R.J. Elder] (ANIC); 1&male; Maalan [17.61S, 145.59E], 3.x.1967, R.J. Elder (ANIC); 2&male;, Tableland [Atherton Tableland], 17.i.1931 (QDAF); 3&male;, Cairns (ANIC). NO LOCALITY: 2&male; (ANIC). Diagnosis. Male. Body 19–22 mm long; bright red-brown (Fig. 15). Terminal palpomere of maxillary palp large, 1.2 mm long, very elongate (length:width 4.5:1), with an elongate, flat, dull, elliptical depression on the latero-dorsal side. Clypeus with anterior face moderately shallow, width 7.5x length; upper surface strongly transverse, width 3.8– 4.0x length, anterior margin indented in middle and margin slightly reflexed to reveal labrum, with broadly rounded angles; upper surface concave, densely punctured and uniformly punctured (approximately 50 mm-2) except just behind reflexed anterior margin, each of the broad, flat-bottomed punctures with a flattened, white seta about as long as radius of the puncture. Frons with similar punctures, sparser in front and denser behind, each puncture with a short, white seta as long as the radius of the puncture, a few punctures in anterior angles with longer, flattened, white setae (2–4x radius of the puncture); posterior edge with only a few punctures and setae; laterally with a few recumbent, long, whiteyellow setae above each eye. Antennae with 10 antennomeres, antennomere 3 elongate-cylindrical, antennomere 4 discoidal, antennomere 5 discoidal but with a faint lump near proximal edge, antennomeres 6 and 7 each with a very short indication of a lamella, antennomeres 8–10 lamellate, 2.2 mm long. Pronotum transverse, greatest width 1.6x length; anterior margin narrow, continuous and raised, punctate except in middle; lateral edges with rounded, obtuse angle in middle, straight before and after angle; posterior margin with narrow, raised margin in middle and towards lateral angles, margin between these with flattened white, short setae; anterior and posterior angles slightly obtuse; surface uniformly punctured (approximately 17 mm-2), each puncture with a minute, slightly elongate, white seta about as long as diameter of the puncture, punctures denser towards anterior angles. Scutellum with line of small punctures close to lateral edge but absent from apex, disc sparsely punctured with setae as on pronotum. Elytra sparsely but fairly uniformly punctured (approximately 10 mm-2) except on sutural interval, each puncture very shallow and with a stout, acutely pointed, white seta as long as those on pronotum. Propygidium uniformly punctured, each puncture with an elongate, short seta. Pygidium irregularly punctured, setae stouter than on propygidium, surface between punctures smooth to microreticulate, long, yellow setae on posterior-lateral margins. Ventral surface of thorax densely clothed with long, fine, yellow setae; pronotal hypemeron with elongate, white setae over all of surface interspersed with a few, long, fine, yellow setae. Teeth on outer edge of protibia moderately long and acute. Ventrites densely clothed with short setae similar to pygidium, less dense on posterior ventrites; first visible ventrite with longer, yellow setae; penultimate ventrite with a few, scattered, long, yellow setae near posterior margin; terminal ventrite without lateral lobes. Aedeagus slightly asymmetrical, distal inner margin of one paramere crossing over the other (Fig. 21). Female. With a less conspicuous flattened area at the end of the last sclerite (Arrow 1932). Distribution (Fig. 25). Known only from near Atherton (Britton 1978). Britton’s (1978) reference to the type locality as Mareeba is obviously incorrect—Mareeba is on a very different soil type and is much drier. Britton (1978) also referred to a specimen from Pialba [25.28°S, 152.84°E] in SAM that I suspect was a misidentified Lepidiota noxia Britton, 1985 (Allsopp 1989), and three specimens from ‘Cairns’ [his use of inverted commas] in the Oslo Museum [presumably, Naturhistorisk Museum, now in ANIC]—Cairns was commonly used for localities to the west of that city. QDAF also has a male labelled ‘Ayr 26.xii.59 GWS’ and a female labelled ‘Eungella, 29.xi. 31W. A. McDougall’. Both are well outside the usual range of the species, and I suspect are mislabelled. Natural history. Adults have been collected in oil-bath traps and at light during December–February (see above records). There are also records from June and October (Arrow 1932; Smith 1936; Britton 1978) that may refer to the collection of larvae. It has a two-year lifecycle (Smith 1936) and is a pest of pastures in areas of alkaline soils near Atherton (Arrow 1932; Smith 1936; Atherton 1939; Cumpston 1941). It is locally referred to as paspalum whitegrub. Gough and Brown (1988) discussed the insecticidal control of the larvae., Published as part of Allsopp, Peter G., 2022, Australian Melolonthini (Coleoptera: Scarabaeidae: Melolonthinae): reclassification of eight species to Antitrogus Burmeister, 1855 and Barryfilius new genus, pp. 513-545 in Zootaxa 5213 (5) on pages 525-527, DOI: 10.11646/zootaxa.5213.5.3, http://zenodo.org/record/7381670, {"references":["Arrow, G. J. (1932) A few new species of melolonthine Coleoptera. Annals and Magazine of Natural History, Series 10, 9, 189 - 197. https: // doi. org / 10.1080 / 00222933208673490","Britton, E. B. (1978) A revision of the Australian chafers (Coleoptera: Scarabaeidae: Melolonthinae). Vol. 2. Tribe Melolonthini. Australian Journal of Zoology, Supplementary Series, 60, 1 - 150. https: // doi. org / 10.1071 / AJZS 060","Allsopp, P. G. (2020 b) Clarification of the status of the types of Australian Melolonthini (Coleoptera: Scarabaeidae: Melolonthinae) described before 1950. Zootaxa, 4885 (4), 451 - 486. https: // doi. org / 10.11646 / zootaxa. 4885.4.1","Britton, E. B. (1985) Lepidiota noxia sp. n. (Coleoptera: Scarabaeidae: Melolonthinae), a pest of sugarcane in Queensland. Journal of the Australian Entomological Society, 24, 117 - 119. https: // doi. org / 10.1111 / j. 1440 - 6055.1985. tb 00202. x","Allsopp, P. G. (1989) Two new species of Lepidiota Kirby (Coleoptera: Scarabaeidae: Melolonthinae) from Australia with notes on L. noxia Britton. Journal of the Australian Entomological Society, 28, 39 - 43. https: // doi. org / 10.1111 / j. 1440 - 6055.1989. tb 01190. x","Smith, J. H. (1936) White grub damage to pastures on the Atherton Tableland. Queensland Agricultural Journal, 46, 446 - 467.","Atherton, D. O. (1939) White grubs and pasture deterioration on the Atherton Tableland. Queensland Agricultural Journal, 52, 484 - 522.","Cumpston, D. M. (1941) A summary of certain aspects of the scarab problem, and a contribution to a bibliography of the family Scarabaeidae. Proceedings of the Linnean Society of New South Wales, 66, 33 - 40.","Gough, N. & Brown, J. D. (1988) Insecticidal control of whitegrubs (Coleoptera: Scarabaeidae) on the Atherton Tableland, with observations on crop losses. Queensland Journal of Agricultural and Animal Sciences, 45, 9 - 17."]}

Barryfilius contiguus (Britton, 1978), new combination (Figs. 14, 20, 25) Lepidiota contigua Britton, 1978: 65, figs. 195–196 (aedeagus), fig. 282A (head and pronotum). Type series. Holotype male in NHML: Australia (examined). Other material examined. AUSTRALIA: Queensland: 1&male;, paratype (ANIC); 1&male;, Windsor Tableland, via Mt Carbine [16.45°S, 145.25°E], 12.i.1980, R.I. Storey; 1&male;, Windsor Tableland, via Mt Carbine [16.45°S, 145.25°E], 27–28.i.1980, R.I. Storey and N. Gough; 13&male;, Windsor Tableland, via Mt Carbine [16.45°S, 145.25°E], 26.xii.1983 – 24.i.1984, Storey and Halfpapp (QDAF, QM); 85&male;, Windsor Tableland, 27.xii.1988 – 10.i.1990, E. Schmidt and ANZSES (QM). Diagnosis. Male. Body 18–20 mm long; uniformly red-brown (Fig. 14). Terminal palpomere of maxillary palp about 0.8 mm long, elongate-elliptical (length:width 2.7:1.0), with a flat, reticulate, elliptical depression on the dorsal side. Clypeus with anterior face moderately deep, width 6x length; upper surface moderately transverse, width 3.2x length, anterior margin slightly indented in middle, with rounded angles; upper surface moderately concave, densely punctured (50 mm-2), each of the broad, flat-bottomed punctures with a flattened, white seta that is shorter than the radius of the puncture. Frons similarly punctured to upper surface of clypeus; posterior quarter with only a few punctures and setae; antero-laterally rarely with a few, recumbent, yellow setae above the eye. Antennae with 10 antennomeres, antennomere 3 elongate-cylindrical, antennomere 4 discoidal, antennomere 5 slightly lamellate, antennomere 6 lamellate but lamella 0.45x long as lamellae of antennomeres 8–10, antennomere 7 lamellate but lamella 0.75x long as lamellae of antennomeres 8–10, antennomeres 8–10 lamellate, 2.4–2.5 mm long. Pronotum transverse, greatest width 1.5–1.6x length; anterior margin narrow, continuous and raised, punctate except in middle; lateral edges with a broadly rounded, obtuse angle in middle, straight before and after angle; posterior margin with a narrow, raised margin in middle; anterior angles slightly obtuse, posterior angles slightly more obtuse; surface of disc uniformly punctured (approximately 17 mm-2), each puncture with a small rounded or slightly elongate, white seta about as long as diameter of the puncture, punctures and setae denser and setae more elongate along posterior margin and lateral margins. Scutellum with a line of small punctures close to lateral edge, disc sparsely punctured with setae as on pronotum. Elytra sparsely but fairly uniformly punctured (ca 10 mm-2) except on sutural interval, each puncture with an elliptical, white seta (0.06 mm long), longer and thinner than those on pronotum. Propygidium with a few punctures with elliptical, white-yellow setae in posterior-lateral angles. Pygidium irregularly punctured in middle, denser towards anterior margin; setae similar to those on elytra, surface between punctures microreticulate, long, yellow setae on posterior-lateral margins. Ventral surface of thorax densely clothed with long, fine, yellow setae; pronotal hypemeron with a mixture of long, yellow setae on inner edge, moderately long, white setae, and broader, shorter, white setae. Teeth on outer edge of protibia short and blunt. Ventrites sparsely clothed with short, setae similar to elytra; first visible ventrite with longer, yellow setae; penultimate ventrite with a few, scattered, long, yellow setae along posterior margin; terminal ventrite without lateral lobes. Aedeagus slightly asymmetrical, apices recurved and close to each other (Fig. 20). Female. Unknown. Distribution (Fig. 25). Britton (1978) saw specimens labelled, at best, ‘Queensland’. The species occurs on the Windsor Tableland, 42 km northwest of Mossman in an isolated area of closed forest described as simple microphyll vine-fern forest (Storey & Allsopp 1989). Natural history. Adults have been collected in December–January in malaise and flight-intercept traps; flying adults were not attracted to lights (Storey & Allsopp 1989)., Published as part of Allsopp, Peter G., 2022, Australian Melolonthini (Coleoptera: Scarabaeidae: Melolonthinae): reclassification of eight species to Antitrogus Burmeister, 1855 and Barryfilius new genus, pp. 513-545 in Zootaxa 5213 (5) on page 525, DOI: 10.11646/zootaxa.5213.5.3, http://zenodo.org/record/7381670, {"references":["Britton, E. B. (1978) A revision of the Australian chafers (Coleoptera: Scarabaeidae: Melolonthinae). Vol. 2. Tribe Melolonthini. Australian Journal of Zoology, Supplementary Series, 60, 1 - 150. https: // doi. org / 10.1071 / AJZS 060","Storey, R. I. & Allsopp, P. G. (1989) A positive locality record for Lepidiota contigua Britton (Coleoptera: Scarabaeidae: Melolonthinae). Australian Entomological Magazine, 16, 47."]}

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.Abstract Figure

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine–metallo enzyme type.

As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea‐type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn‐glycerol 1‐phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O‐acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first‐time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects.

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme’s co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

Background and objective: Antibiotics are widely used worldwide. However, due to the emergence of antibiotic resistance in a wide range of microorganisms, their use worldwide has failed. Probiotics are suggested as complementary and alternative medicine. The present study aimed to investigate the effects of probiotics isolated from local chickens on the expression of luxS and ctxM genes in resistant Escherichia coli. Materials and methods: 300 fecal samples were taken from patients referring to Imam Khomeini Hospital in Tehran during May–September 2016 and Escherichia coli samples were isolated using specific culture media and biochemical tests and then the presence of luxS and ctxM genes were identified using PCR with specific primers. In order to extract the probiotics forming spores, the intestinal contents of 10 poultry that had not used any antibiotics and probiotics were cultured, isolated, and identified using biochemical and PCR methods. Commercial strains of Bacillus subtilis and Bacillus coagulans were purchased to compare their effects with native bacteria. These strains were then co-cultured with resistant Escherichia coli strains containing ctxM and luxS genes. Real-time PCR was used to evaluate the effect of these probiotics on gene expression Results: The results indicated that 40 isolates (7.5%) of Escherichia coli were obtained from the 300 fecal samples. Thirteen samples (32.5%) were outpatients and 27 (67.5%) were inpatients. All isolates were isolated from men and women aged 21–62. Four Escherichia coli strains were isolated from patients carrying ctxM and luxS genes. Isolation of Bacillus coagulans and Bacillus subtilis from samples was confirmed by biochemical and molecular experiments. The commercial and native strains of Bacillus coagulans reduced the expression of the luxS and ctxM genes by 3.60, 3.30, 1.58, and 2.70 times respectively. Also, the commercial and native strains of Bacillus subtilis decreased the expression of the luxS and ctxM genes by 1.37, 1.10, 2.20, and 2.80 times respectively. The results of statistical analysis showed a significant relationship between the presence of native and commercial probiotics in culture and reduced expression of ctxM and luxS genes. Conclusion: According to the results, supplements of Bacillus coagulans and Bacillus subtilis increase the effect of antibiotics resistance in Escherichia coli by reducing the expression of resistance genes.

The aerobic endospore-forming bacteria (AEFB) comprise species of Bacillus and related genera, allocated in the phylum Firmicutes. Although Bacillus spp. are among the first bacteria to be characterised, the wide diversity renders appropriate categorisation and generalisations challenging tasks. To determine genetic diversity, analyses at the molecular level are the most accurate. However, gene expression, morphological, biochemical, and physiological aspects must also be considered. The metabolism of bacteria is adapted to their natural environment or host. Thus, metabolic outlines can be used for identifying AEFB, form the basis of the formal description of bacterial taxa, and are strongly recommended for taxonomic purposes. This work addressed the biochemical and physiological profiles of 312 environmental AEFB—designated as SDF (Solo do Distrito Federal)—by performing 30 tests. Out of it, 246 were classified by 16S rRNA gene sequences. We summarised the phenotypic test relationships among selected SDF strains using a Pearson correlation-based clustering represented in heatmaps. In practice, biochemical and physiological profiles are often less discriminatory than molecular data and may be unstable because of the loss of traits. Though these test reactions are not universally positive or negative within species, they may define biotypes and be efficient strain markers, enhancing the accuracy of unknown sample identification. It can be also helpful in selecting the best represent the phenotypes of samples. Along with the other phenotypic and genotypic data, the present results will be of great importance for the robust classification of the SDF strains within the scope of the polyphasic approach.

Stereoselective synthesis of d-glycero- and l-glycero-β-d-mannoheptosides has been achieved by cesium carbonate-mediated β-selective anomeric O-alkylation of the corresponding d-mannoheptoses. In addition, this method has been utilized in the total synthesis of a tetrasaccharide repeat unit of Bacillus thermoaerophilus surface-layer glycoprotein.

Background The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. Results Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. Conclusions Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium.

Aneurinibacillus thermoaerophilus CCM 8960 is a thermophilic bacterium isolated from compost in Brno. The bacterium accumulates polyhydroxyalkanoates (PHAs), a biodegradable and renewable alternative to petrochemical polymers. The bacterium reveals several features that make it a very interesting candidate for the industrial production of PHA. At first, due to its thermophilic character, the bacterium can be utilized in agreement with the concept of next-generation industrial biotechnology (NGIB), which relies on extremophiles. Second, the bacterium is capable of producing PHA copolymers containing a very high portion of 4-hydroxybutyrate (4HB). Such materials possess unique properties and can be advantageously used in multiple applications, including but not limited to medicine and healthcare. Therefore, this work focuses on the in-depth characterization of A. thermoaerophilus CCM 8960. In particular, we sequenced and assembled the genome of the bacterium and identified its most important genetic features, such as the presence of plasmids, prophages, CRISPR arrays, antibiotic-resistant genes, and restriction-modification (R-M) systems, which might be crucial for the development of genome editing tools. Furthermore, we focused on genes directly involved in PHA metabolism. We also experimentally studied the kinetics of glycerol and 1,4-butanediol (1,4BD) utilization as well as biomass growth and PHA production during cultivation. Based on these data, we constructed a metabolic model to reveal metabolic fluxes and nodes of glycerol and 1,4BD concerning their incorporation into the poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) structure. KEY POINTS: • Aneurinibacillus sp. H1 was identified as Aneurinibacillus thermoaerophilus. • PHA metabolism pathway with associated genes was presented. • Unique monomer composition of produced PHAs was reported.

Xylan and pectin are major structural components of plant cell walls. There are two independent catabolic pathways for xylan and pectin. UxaE bridges these two pathways by reversibly epimerizing D-fructuronate and D-tagaturonate. The crystal structure of UxaE from Cohnella laeviribosi (ClUxaE) shows a core scaffold of TIM-barrel with a position-changing divalent metal cofactor. ClUxaE has the flexible metal-coordination loop to allow the metal shift and the extra domains to bind a phosphate ion in the active site, which are important for catalysis and substrate specificity. Elucidation of the structure and mechanism of ClUxaE will assist in understanding the catalytic mechanism of UxaE family members, which are useful for processing both xylan and pectin-derived carbohydrates for practical and industrial purposes, including the transformation of agricultural wastes into numerous valuable products.

Membrane vesicles (MVs) released from bacteria act as extracellular vehicles carrying various functional cargoes between cells. MVs with different cargoes play multiple roles in stress adaptation, nutrient acquisition and microbial interactions. However, previous studies have primarily focused on MVs from Gram-negative bacteria, while the characteristics of cargoes in MVs from Gram-positive bacteria and their involvement in microbial interactions remain to be elucidated. Here, we used a Gram-positive strain, Dietzia sp. DQ12-45-1b from Corynebacteriales, to analyse the characteristics and functions of MVs. We identified the 'antioxidant' canthaxanthin is stored within MVs by LC-MS/MS. In addition, nearly the entire genomic content of strain DQ12-45-1b are evenly distributed in MVs, suggesting that MVs from DQ12-45-1b might involve in horizontal gene transfer. Finally, the mycobactin-type siderophores were detected in MVs. The iron-loaded MVs effectively mediate iron binding and delivery to homologous bacteria from the order Corynebacteriales, but not to more distantly related species from the orders Pseudomonadales, Bacillales and Enterobacterales. These results revealed that the iron-loaded MVs are shared between homologous species. Together, we report the Gram-positive bacterium Dietzia sp. DQ12-45-1b released MVs that contain canthaxanthin, DNA and siderophores and prove that MVs act as public goods between closely related species.

Fermentation of carbohydrates present in lignocellulosic (LC) biomass is facilitated by lignin removal, which is usually achieved by adopting various pretreatment methods to provide the enzymes proper access to their respective substrates. Pretreatment using ionic liquid (IL) is relatively recent advancement and considered as mild and green process. ILs can dissolve extensive quantities of biomass and depolymerize the cellulose. In this context, an abundantly available LC biomass, sugarcane bagasse (SB), was pretreated using alkali or with an IL, methyltrioctylammonium chloride, and was used for cellulase production from thermophilic bacteria. In all, 26 indigenously isolated thermophilic bacterial strains were quantitatively screened for cellulase production. 16S rDNA sequences of the promising isolates UE10 and UE27 revealed relatedness with Brevibacillus borstelensis, while the strain UE1 belonged to Aneurinibacillus thermoaerophilus. Cellulase production was compared by utilizing alkali pretreated and IL pretreated SB and the later was found more appropriate. UE1, UE10 and UE27 yielded 22.2, 22.18 and 33.3 IU mL-1 of endoglucanase, respectively, by fermenting IL pretreated SB. The changes in SB structure after pretreatment were evaluated by scanning electron microscopy. This study demonstrated the potential of novel thermophilic bacterial strains to utilize IL pretreated SB for production of industrially important enzyme, cellulase.

A total of 262 bacterial strains were isolated from clay minerals, bentonite and zeolite, in Gyeongsangbukdo, Republic of Korea, and their hydrolytic enzyme activities were analyzed. Most of the isolated strains belonged to Micrococcales and Bacillales order. Of strains, 96 strains produced α-amylase activity, 42 strains showed cellulase activity, 111 strains had pectinase activity, and 70 strains showed protease activity. Among them, 177 isolates exhibited one or more of the hydrolytic enzyme activities and in particular Bacillus cereus MBLB1321, B. albus MBLB1326 and KIGAM017, B. mobilis MBLB1328, MBLB1329 and MBLB1330 showed all of the enzyme activities. These results demonstrate the diversity of functional Bacillus species in clay minerals as vital sources for the discovery of industrially valuable hydrolytic enzymes, which have a great commercial prospect in various bio-industrial applications.

Inactivation of Bacillales and Clostridiales spores is of interest, since some cause food spoilage and human diseases. A recent publication (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of Bacillus subtilis, Bacillus cereus and Clostridioides difficile, and Bacillus anthracis spores to a lesser extent. We now show that (i) the B. subtilis spores prepared as in the prior work were impure; (ii) if spore viability was measured by diluting spores 1/10 in GMLg, serially diluting incubations 10-fold and spotting aliquots on recovery plates, there was no colony formation from the 1/10 to 1/1000 dilutions due to GMLg carryover, although thorough ethanol washes of incubated spores eliminated this problem and (iii) GMLg did not kill highly purified spores of B. subtilis, B. cereus, Bacillus megaterium and C. difficile in 3-20 h in the conditions used in the recent publication. GMLg also gave no killing of crude B. subtilis spores prepared as in the recent publication in 5 h but gave ~1·5 log killing at 24 h. Thus, GMLg does not appear to be an effective sporicide, although the gel likely inhibits spore germination and could kill spores somewhat upon long incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: Given potential deleterious effects of spores of Bacillales and Clostridiales, there is an ongoing interest in new ways of spore killing. A recent paper (mSphere 3: e00597-1, 2018) reported that glycerol monolaurate (GML) in a non-aqueous gel (GMLg) effectively killed spores of many species. We now find that (i) the Bacillus subtilis spores prepared as in the previous report were impure and (ii) GMLg gave no killing of purified spores of Bacillales and Clostridiales species in ≤5 h under the published conditions. Thus, GMLg is not an effective sporicide, though may prevent spore germination or kill germinated spores.

Polyhydroxyalkanoates are microbial polyesters which are considered being biological alternatives to petrochemical polymers. Extremophiles, such as thermophilic PHA producers, hold promise to improve competitiveness of PHA production process. Therefore, this work aimed at isolation of new strains, which could produce PHA under elevated temperature. Since traditional Nile red staining of colonies provided false positive results in thermophiles, we developed a novel strategy of enriching microbial consortia by PHA producers. This so called "osmoselective strategy" is based on application of osmotic challenge by sudden exposition of the mixed microbial culture to hypertonic and subsequently to hypotonic conditions; moreover, this strategy relies on the fact that PHA protect bacteria from negative effects of rapid fluctuations in osmotic pressure. In combination with fast and reliable ATR-FTIR inspection of selected colonies for presence of PHA, we were able to isolate several promising thermophilic or thermotolerant PHA producing strains belonging to the genera Bacillus, Aneurinibacillus and Chelatococcus, which indeed deserves further investigation to evaluate their potential for industrial production of PHA.

The study was envisioned to evaluate the decolorization of Reactive Blue 160 (RB160) dye by using indigenous microbes. Contaminated soil from textile dye industry was collected from Noyyal river basin, Tamil Nadu, India. Potential dye degrading bacterial strain was recognized as Bacillus firmus by 16SrRNA gene sequencing analysis. RB160 dye (500 μg/ml) was effectively degraded by B. firmus and toxicological analyses were performed with RB160 and their degraded product. Phytotoxicity revealed that degraded product of RB160 into non-toxic nature by B. firms. Toxicity assays were carried out on root cells of Allium cepa and human skin cell line (CRL 1474). Toxicity analysis of A. cepa and cell line signifies that dye exerts toxic cause on the root cells and IC50 values of RB160 showed toxic to human skin cell lines, while degradation products of the dye are moderately less in toxic. Zebrafish embryo toxicity also evaluated by RB160 and degraded product on phenotypic deformation, survival, hatching and heartbeat rate. However, RB160 with concentration of 500 μg/ml decrease in the survival, hatching, heartbeat rate and induced phenotypic alterations. In which, degraded products exhibited significant development in zebrafish embryos as compared to dye. Based on the studies effects of RB160 and capability of B. firmus can effectively degrade RB160, and their degraded products were harmless to the environments and aquatic system.

Biogenic coalbed methane is produced by biological processes mediated by synergistic interactions of microbial complexes in coal seams. However, the ecological role of functional bacteria in biogenic coalbed methane remains poorly understood. Here, we studied the metagenome assembled genomes (MAGs) of Bacillales and Clostridiales from coal seams, revealing further expansion of hydrogen and acetogen producers involved in organic matter decomposition. In this study, Bacillales and Clostridiales were dominant orders (91.85 ± 0.94%) in cultured coal seams, and a total of 16 MAGs from six families, including Bacillus, Paenibacillus, Staphylococcus, Anaerosalibacter, Hungatella and Paeniclostridium, were reconstructed. These microbial groups possessed multiple metabolic pathways (glycolysis/gluconeogenesis, pentose phosphate, β-oxidation, TCA cycle, assimilatory sulfate reduction, nitrogen metabolism and encoding hydrogenase) that provided metabolic substrates (acetate and/or H2) for the methanogenic processes. Therein, the hydrogenase-encoding gene and hydrogenase maturation factors were merely found in all the Clostridiales MAGs. β-oxidation was the main metabolic pathway involved in short-chain fatty acid degradation and acetate production, and most of these pathways were detected and exhibited different operon structures in Bacillales MAGs. In addition, assimilatory sulfate reduction and nitrogen metabolism processes were also detected in some MAGs, and these processes were also closely related to acetate production and/or organic matter degradation according to their operon structures and metabolic pathways. In summary, this study enabled a better understanding of the ecological roles of Bacillales and Clostridiales in biogenic methane in coal seams based on a combination of bioinformatic techniques.

L-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

Traditionally, starting inoculants have been applied to improve ensiling of forage used for livestock feed. Here, we aimed to build up a bioinoculant composed of lactic acid-producing and lignocellulolytic bacteria (LB) derived from the Megathyrsus maximus (guinea grass) phyllosphere. For this, the dilution-to-stimulation approach was used, including a sequential modification of the starting culture medium [Man, Rogosa, and Sharpe (MRS) broth] by addition of plant biomass (PB) and elimination of labile carbon sources. Along 10 growth-dilution steps (T1–T10), slight differences were observed in terms of bacterial diversity and composition. After the sixth subculture, the consortium started to degrade PB, decreasing its growth rate. The co-existence of Enterobacteriales (fast growers and highly abundance), Actinomycetales, Bacillales, and Lactobacillales species was observed at the end of the selection process. However, a significant structural change was noticed when the mixed consortium was cultivated in higher volume (500ml) for 8days, mainly increasing the proportion of Paenibacillaceae populations. Interestingly, Actinomycetales, Bacillales, and Lactobacillales respond positively to a pH decrease (4–5), suggesting a relevant role within a further silage process. Moreover, gene-centric metagenomic analysis showed an increase of (hemi)cellulose-degrading enzymes (HDEs) during the enrichment strategy. Reconstruction of metagenome-assembled genomes (MAGs) revealed that Paenibacillus, Cellulosimicrobium, and Sphingomonas appear as key (hemi)cellulolytic members (harboring endo-glucanases/xylanases, arabinofuranosidases, and esterases), whereas Enterococcus and Cellulosimicrobium have the potential to degrade oligosaccharides, metabolize xylose and might produce lactic acid through the phosphoketolase (PK) pathway. Based on this evidence, we conclude that our innovative top-down strategy enriched a unique bacterial consortium that could be useful in biotechnological applications, including the development/design of a synthetic bioinoculant to improve silage processes.

Bacteria from the orders Bacillales and Clostridiales differentiate into stress-resistant spores that can remain dormant for years, yet rapidly germinate upon nutrient sensing. How spores monitor nutrients is poorly understood but in most cases requires putative membrane receptors. The prototypical receptor from Bacillus subtilis consists of three proteins (GerAA, GerAB, GerAC) required for germination in response to L-alanine. GerAB belongs to the Amino Acid-Polyamine-Organocation superfamily of transporters. Using evolutionary co-variation analysis, we provide evidence that GerAB adopts a structure similar to an L-alanine transporter from this superfamily. We show that mutations in gerAB predicted to disrupt the ligand-binding pocket impair germination, while mutations predicted to function in L-alanine recognition enable spores to respond to L-leucine or L-serine. Finally, substitutions of bulkier residues at these positions cause constitutive germination. These data suggest that GerAB is the L-alanine sensor and that B subunits in this broadly conserved family function in nutrient detection., Germination of Bacillus subtilis spores in response to L-alanine requires a putative membrane receptor consisting of three proteins. Here, Artzi et al. use evolutionary co-variation analysis and functional assays of mutants to provide evidence that one of the proteins, GerAB, likely acts as the L-alanine sensor.

Heat shock-tolerant microorganisms belonging to the orders Bacillales and Micrococcales were isolated from the Spacecraft Assembly Facility at the Jet Propulsion Laboratory, and 63 draft genome sequences were assembled and identified. Further analyses of these genomes can provide insight into methods for preventing forward contamination.

The bacterial microbiota in the skin and intestine of patients with psoriasis were different compared with that of healthy individuals. However, the presence of a distinct blood microbiome in patients with psoriasis is yet to be investigated. In this study, we investigated the differences in bacterial communities in plasma-derived extracellular vesicles (EVs) between patients with moderate to severe psoriasis (PSOs) and healthy controls (HCs). The plasma EVs from the PSO (PASI &gt, 10) (n = 20) and HC (n = 8) groups were obtained via a series of centrifugations, and patterns were examined and confirmed using transmission electron microscopy (TEM) and EV-specific markers. The taxonomic composition of the microbiota was determined by using full-length 16S ribosomal RNA gene sequencing. The PSO group had lower bacterial diversity and richness compared with HC group. Principal coordinate analysis (PCoA)-based clustering was used to assess diversity and validated dysbiosis for both groups. Differences at the level of amplicon sequence variant (ASV) were observed, suggesting alterations in specific ASVs according to health conditions. The HC group had higher levels of the phylum Firmicutes and Fusobacteria than in the PSO group. The order Lactobacillales, family Brucellaceae, genera Streptococcus, and species Kingella oralis and Aquabacterium parvum were highly abundant in the HC group compared with the PSO group. Conversely, the order Bacillales and the genera Staphylococcus and Sphihgomonas, as well as Ralstonia insidiosa, were more abundant in the PSO group. We further predicted the microbiota functional capacities, which revealed significant differences between the PSO and HC groups. In addition to previous studies on microbiome changes in the skin and gut, we demonstrated compositional differences in the microbe-derived EVs in the plasma of PSO patients. Plasma EVs could be an indicator for assessing the composition of the microbiome of PSO patients.

For a long time, the genus Bacillus has been known and considered among the most applicable genera in several fields. Recent taxonomical developments resulted in the identification of more species in Bacillus-related genera, particularly in the order Bacillales (earlier heterotypic synonym: Caryophanales), with potential application for biotechnological and industrial purposes such as biofuels, bioactive agents, biopolymers, and enzymes. Therefore, a thorough understanding of the taxonomy, growth requirements and physiology, genomics, and metabolic pathways in the highly diverse bacterial order, Bacillales, will facilitate a more robust designing and sustainable production of strain lines relevant to a circular economy. This paper is focused principally on less-known genera and their potential in the order Bacillales for promising applications in the industry and addresses the taxonomical complexities of this order. Moreover, it emphasizes the biotechnological usage of some engineered strains of the order Bacillales. The elucidation of novel taxa, their metabolic pathways, and growth conditions would make it possible to drive industrial processes toward an upgraded functionality based on the microbial nature.

Aerobic spore-forming Bacillales are a highly diverse and ubiquitous group that includes organisms that cause foodborne illnesses and food spoilage. Classical microbiological and biochemical identification of members of the order Bacillales represents a challenge due to the diversity of organisms in this group as well as the fact that the phenotypic-based taxonomic assignment of some named species in this group is not consistent with their phylogenomic characteristics. DNA-sequencing-based tools, on the other hand, can be fast and cost-effective, and can provide for a more reliable identification and characterization of Bacillales isolates. In comparison to 16S rDNA , rpoB was shown to better discriminate between Bacillales isolates and to allow for improved taxonomic assignment to the species level. However, the lack of a publicly accessible rpoB database, as well as the lack of standardized protocols for rpoB-based typing and strain identification, is a major challenge. Here, we report (i) the curation of a DNA sequence database for rpoB-based subtype classification of Bacillales isolates; (ii) the development of standardized protocols for generating rpoB sequence data, and a scheme for rpoB- based initial taxonomic identification of Bacillales isolates at the species level; and (iii) the integration of the database in a publicly accessible online platform that allows for the analysis of rpoB sequence data from uncharacterized Bacillales isolates. Specifically, we curated a database of DNA sequences for a 632-nt internal variable region within the rpoB gene from representative Bacillales reference type strains and a large number of isolates that we have previously isolated and characterized through multiple projects. As of May 21, 2021, the rpoB database contained more than 8350 rpoB sequences representing 1902 distinct rpoB allelic types that can be classified into 160 different genera. The database also includes 1129 rpoB sequences for representative Bacillales reference type strains as available on May 21, 2021 in the NCBI database. The rpoB database is integrated into the online Food Microbe Tracker platform ( www.foodmicrobetracker.com ) and can be queried using the integrated BLAST tool to initially subtype and taxonomically identify aerobic and facultative anaerobic spore-formers. While whole-genome sequencing is increasingly used in bacterial taxonomy , the rpoB sequence-based identification scheme described here provides a valuable tool as it allows for rapid and cost-effective initial isolate characterization, which can help to identify and characterize foodborne pathogens and food spoilage bacteria. In addition, the database and primers described here can also be adopted for metagenomics approaches that include rpoB as a target, improving discriminatory power and identification over what can be achieved using 16S rDNA as a target.

Bacillus thuringiensis (Bt) (Bacillales:Bacillaceae) is a gram-positive bacterium that produces spores, several virulence factors and insecticidal toxins, making this microorganism the most used biopesticide worldwide. The use of inert supports such as polyurethane foam (PUF) in solid cultures has been a great alternative to produce various metabolites, including those produced by Bt. In this study we compared the yields, productivity and quality of the spores by two wild strains of Bt, (Y15 and EA3), grown in media with high substrate concentration in both culture systems: liquid and solid (PUF as solid inert support). Both strains showed 2.5- to 30-fold increases in spore production and productivity in solid culture, which showed an even greater increase when considering the spores retained in the PUF observed by scanning electron microscopy. Moreover, spore produced in solid culture showed up to sevenfold higher survival after a heat-shock treatment, relative to spores from liquid culture. The infectivity against larvae of Galleria mellonella (Lepidoptera:Pyralidae) improved also in spores from solid cultures. This comparison showed that the culture of Bt on solid support has clear advantages over liquid culture in terms of the production and quality of spores, and that those advantages can be attributed only to the culture system, as the same media composition was used in both systems.

Paludifilum halophilum DSM 102817

Spores of many species of the orders Bacillales and Clostridiales can be vectors for food spoilage, human diseases and intoxications, and biological warfare. Many agents are used for spore killing, including moist heat in an autoclave, dry heat at elevated temperatures, UV radiation at 254 and more recently 222 and 400 nm, ionizing radiation of various types, high hydrostatic pressures and a host of chemical decontaminants. An alternative strategy is to trigger spore germination, as germinated spores are much easier to kill than the highly resistant dormant spores-the so called "germinate to eradicate" strategy. Factors important to consider in choosing methods for spore killing include the: (1) cost; (2) killing efficacy and kinetics; (3) ability to decontaminate large areas in buildings or outside; and (4) compatibility of killing regimens with the: (i) presence of people; (ii) food quality; (iii) presence of significant amounts of organic matter; and (iv) minimal damage to equipment in the decontamination zone. This review will summarize research on spore killing and point out some common flaws which can make results from spore killing research questionable.

Cellulases are hydrolytic enzymes with wide scientific and industrial applications. We described a novel cellulase, CelC307, from the thermophilic indigenous Cohnella sp. A01. The 3-D structure of the CelC307 was predicted by comparative modeling. Docking of CelC307 with specific inhibitors and molecular dynamic (MD) simulation revealed that these ligands bound in a non-competitive manner. The CelC307 protein was purified and characterized after recombinant expression in Escherichia coli (E. coli) BL21. Using CMC 1% as the substrate, the thermodynamic values were determined as Km 0.46 mM, kcat 104.30 × 10–3 (S−1), and kcat/Km 226.73 (M−1 S−1). The CelC307 was optimally active at 40 °C and pH 7.0. The culture condition was optimized for improved CelC307 expression using Plackett–Burman and Box–Behnken design as follows: temperature 20 °C, pH 7.5, and inoculation concentration with an OD600 = 1. The endoglucanase activity was positively modulated in the presence of Na+, Li+, Ca2+, 2-mercaptoethanol (2-ME), and glycerol. The thermodynamic parameters calculated for CelC307 confirmed its inherent thermostability. The characterized CelC307 may be a suitable candidate for various biotechnological applications.

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.

The screening, identification, and study of the functional properties of cellulolytic xylanolytic bacteria are crucial for the construction of applicable bioprocesses. The thermophilic facultatively anaerobic, xylanolytic bacterial strain DA-C8 (=JCM34211=DSM111723) exhibiting efficient xylan degradation was newly isolated from compost. Strain DA-C8 completely degraded 1% beechwood xylan within 4 days under anaerobic conditions. By 16S rRNA gene sequence homology and phylogenetic tree analysis, strain DA-C8 was closely related to Paenibacillus cisolokensis and Xylanibacillus composti; however, the average nucleotide identity and digital DNA-DNA hybridization values based on genome information and the carbon source utilization properties indicated that strain DA-C8 belongs to Paenibacillus rather than Xylanibacillus. The gene numbers of xylanase and endoglucanase of strain DA-C8 and X. composti were not different; however, strain DA-C8 had higher abundance of α- L -arabinofuranosidase, β-xylosidase, and β-glucosidase than X. composti. Strain DA-C8 showed decreased xylan and corn hull degradation abilities and growth on xylan medium under aerobic conditions. Quantitative PCR showed high expression of xylan and cellulose degradation genes under anaerobic conditions, but the genes were repressed under aerobic conditions, indicating that strain DA-C8 controls polysaccharide degradation depending on the aeration conditions. Strain DA-C8 is a new species of Paenibacillus with a unique polysaccharide degradation system.

A history of drought exposure promoted by variable precipitation regimes can select for drought-tolerant soil microbial taxa, but the mechanisms of survival and death of microbial populations through the selective stresses of soil drying and re-wet are not well understood. We subjected soils collected from a 15-year field drought experiment ("Altered" precipitation history with extended dry periods, versus the "Ambient" field control) to a laboratory drying/re-wetting experiment, to learn whether selective population survival, death, or maintenance of protein synthesis potential and microbial respiration through variable soil water conditions was affected by field drought legacy. Microbial community composition, as measured by Illumina MiSeq sequencing of the 16S rRNA and 16S rRNA gene, shifted with laboratory drying/re-wet and field drought treatments. In Ambient soils, there was a higher proportion of reduced OTU abundance (indicative of mortality) during re-wet, whereas Altered soils had a greater proportion of stable OTU populations that did not change in abundance (indicative of survival) through drying/re-wet. Altered soils also had a lower proportion of rRNA:rRNA genes (lower protein synthesis potential) during dry-down, a greater weighted mean rRNA operon number (potential growth rate and r-selection) which was associated with higher abundance of Firmicutes (order Bacillales), and lower average microbial respiration rates. These data demonstrate that soils with a weaker historical drought legacy exhibit a higher prevalence of microbial water-stress mortality and differential survival and death at OTU levels following short-term dryingand re-wetting, concurrent with higher carbon loss potential. This work provides novel insight into the mechanisms and consequences of soil microbial changes resulting from extended drought conditions.

Daqu starter, an important saccharifying and fermenting agent for the brewing process of traditional vinegar, is manufactured by spontaneous solid-state fermentation which routinely undergoes low or medium incubation temperature. Previous studies have demonstrated that the temperature plays a pivotal role in Daqu quality. Hence, to explore the feasibility of high temperature fermentation applied in the vinegar Daqu brewing and provide guidelines of controlling environmental parameters in traditional vinegar industries, the microbial community and metabolites of vinegar Daqu during medium-temperature and high-temperature fermentation processes (namely, MTFP and HTFP) were compared. The results indicated that the glucoamylase activity, amylase activity and microbial community showed no significant difference in the end of two batches (P > 0.05). Enterobacteriales, Lactobacillales, Bacillales, Saccharomycetales and Mucorales were the dominant orders during MTFP and HTFP. Redundancy analysis revealed that incubation temperature showed positive correlation with the microbial composition from days 3–14 of the fermentation process and was positively associated with the predominant phylotypes of Bacillales, Mucorales, Xanthomonadales and Rickettsiales. The acidity and moisture showed major correlations with microbial composition on day 1 of MTFP and were positively related with the predominant phylotypes of Mucorales and Lactobacillales at the order level. Moreover, higher relative contents of all volatiles were shown in the end of HTFP (13.91 mg/100 g Daqu) compared to MTFP (10.01 mg/100 g Daqu). This work illustrates high temperature (approximately 60°C) fermentation is promising to improve the vinegar Daqu flavor and shall likely contribute to preferably make traditional Daqu by modulating steerable environmental parameters.

A strictly aerobic, motile, endospore-forming, rod-shaped bacterium, designated HS21T, was isolated from rhizospheric soil of the Korean fir tree (Abies koreana) from Halla mountain on Jeju island, Korea. Growth of strain HS21T was observed at pH 6.0-8.0 (optimum: pH 7.0), 0-2% (w/v) NaCl and 4-30°C (optimum: 25°C). A comparative analysis of 16S rRNA gene sequences showed that strain HS21T was most closely related to Cohnella luojiensis HY-22RT (97.6%), followed by C. lupini RLAHU4BT (97.4%) and C. collisoli NKM-5T (97.2%). The genome of strain HS21T comprised a circular chromosome of 7,059,027 bp with 44.8% G + C content. The DNA-DNA relatedness values between strain HS21T and C. luojiensis HY-22RT and C. lupini RLAHU4BT were 18.1% and 13.8%, respectively. The major cellular fatty acids (> 5%) of the isolate were anteiso-C15:0, iso-C16:0, C16:0, and iso-C15:0. The polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysylphosphatidylglycerol, and three unidentified aminophospholipids. Based on its phenotypic, phylogenetic, genomic, and chemotaxonomic properties, strain HS21T represents a novel species of the genus Cohnella, for which the name Cohnella abietis sp. nov. is proposed. The type strain is HS21T (= KCTC 43028T = CCTCC AB 2019010T).