Marinobacterium iners (Iizuka and Komagata 1964) comb. nov. arising from the Synonymy of Marinobacterium georgiense González et al. 1997 and Pseudomonas iners Iizuka and Komagata 1964 (Approved Lists 1980) Abstract Although there is documented evidence in the literature that Marinobacterium georgiense González et al. 1997 and Pseudomonas iners Iizuka and Komagata 1964 (Approved Lists 1980) should be treated as heterotypic synonyms, the nomenclatural consequences have not been implemented. Based on the rules of the International Code of Nomenclature of Prokaryotes when Marinobacterium georgiense González et al. 1997 and Pseudomonas iners Iizuka and Komagata 1964 (Approved Lists 1980) are considered to belong to the genus Marinobacterium González et al. 1997, the earliest epithet (from the competing heterotypic synonyms) is to be used for the resulting taxon, i.e., the combination Marinobacterium iners (Iizuka and Komagata 1964) must be created.

Animal-Use Antibiotics Induce Cross-Resistance in Bacterial Pathogens to Human Therapeutic Antibiotics Abstract Exposure of bacteria to a sub-lethal dosage of antibiotic is one the major causes for the onset of antibiotic resistance. Therefore, we aimed to assess the emergence of antibiotic cross-resistance in bacteria after exposure to a sub-lethal dose of veterinary feed directive (VFD) antibiotics, tilmicosin, and florfenicol. The minimum inhibitory concentrations (MICs) of tilmicosin and florfenicol against Salmonella enterica serovar Enteritidis, Klebsiella pneumoniae, Staphylococcus aureus, and Listeria monocytogenes were determined. Next, the pathogens were exposed to a sub-inhibitory concentration of tilmicosin (0.5, 5, 20 µg/ml) and florfenicol (1, 20 µg/ml) for 24 h and 48 h, and acquired cross-resistance to human therapeutic antibiotics was measured by determining the increase in MIC values. MICs of ampicillin, tetracycline, nalidixic acid, and meropenem against Salmonella and Klebsiella were in the range of 20–1000 µg/ml, 5–62.5 µg/ml, 5–125 µg/ml, and 0.05–0.1 µg/ml, respectively, whereas MICs against Staphylococcusand Listeria were 2.5–10 µg/ml, 2.5 µg/ml, 62.5–500 µg/ml, and 0.1–0.2 µg/ml, respectively. Pre-exposure of these bacteria to a sub-inhibitory concentration of tilmicosin and florfenicol, increased cross-resistance against ampicillin, tetracycline, and nalidixic acid from 1.25- to 40-fold compared to the antibiotic unexposed bacteria with the exception of meropenem, which did not show increased resistance. This study could serve as a foundation to understand the mechanisms of acquired cross-resistance to traditional therapeutic antibiotics, and to develop strategies to alleviate such problem by using alternative antimicrobials.

The Xanthomonas citri pv. citri Type VI Secretion System is Induced During Epiphytic Colonization of Citrus Abstract Xanthomonas citri pv. citri (X. citri pv. citri) is the causal agent of Asiatic citrus canker and infects economically important citrus crops. X. citri pv. citri contains one type VI secretion system (T6SS) required for resistance to predation by the soil amoeba Dictyostelium discoideum and induced by the ECF sigma factor EcfK in the presence of amoeba. In this work, we describe the analysis of T6SS gene expression during interaction with host plants. We show that T6SS genes and the cognate positive regulator ecfK are upregulated during growth in the plant surface (epiphytic) and maintain low expression levels during growth inside plant mesophyll. In addition, expression of the virulence-associated T3SS is also induced during epiphytic growth and shows a temporal induction pattern during growth inside plant leaves. The T6SS is not required for adhesion to leaf surface and biofilm formation during the first stages of plant colonization nor for killing of yeasts cells. Since the phyllosphere is colonized by eukaryotic predators of bacteria, induction of the X. citri pv. citri anti-amoeba T6SS during epiphytic growth suggests the presence of an environmental signal that triggers the resistance phenotype.

Bacillus subtilis Spore Surface Display of Haloalkane Dehalogenase DhaA Abstract The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2′-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilisspores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.

Engineering Lactococcus lactis for D-Lactic Acid Production from Starch Abstract Bioprocess development is a current requirement to enhance the global production of D-lactic acid. Herein, we report a new bioprocess for D-lactic acid production directly from starch using engineered Lactococcus lactis NZ9000. To modify L. lactis as a D-lactic acid producer, its major endogenous L-lactate dehydrogenase (L-Ldh) gene was replaced with a heterologous D-Ldh gene from Lactobacillus delbrueckii subsp. lactis JCM 1107. The resulting strain AH1 showed a somewhat slower growth rate but similar lactic acid production compared to those of the intact strain when cultivated with glucose as a carbon source. The chemical purity of D-lactic acid produced by L. lactis AH1 was 93.8%, and the enzymatic activities of D- and L-Ldh in AH1 were 1.54 U/mL and 0.05 U/mL, respectively. Next, a heterologous α-amylase gene from Streptococcus bovis NRIC 1535 cloned into an expression vector pNZ8048 was introduced into AH1. The resulting strain AH2 showed an amylolytic activity of 0.26 U/mL in the culture supernatant. Direct production of D-lactic acid from starch as the carbon source was demonstrated using L. lactis AH2, resulting in D-lactic acid production at a concentration of 15.0 g/L after 24 h cultivation. To our knowledge, this is the first report on D-lactic acid production in engineered L. lactis.

Lachnoanaerobaculum gingivalis sp. nov., Isolated from Human Subgingival Dental Plaque of a Gingivitis Lesion Abstract A novel Gram-stain-positive, obligately anaerobic, spore-forming rod, designated strain ChDC B114T, was isolated from a human dental plaque of a gingivitis lesion. The strain was characterized by polyphasic taxonomic analysis to identify it at the species level. The 16S ribosomal RNA gene (16S rDNA) sequence analysis revealed that the strain belongs to the genus Lachnoanaerobaculum. The percent similarity of the 16S rDNA of the strain was closest to the homologous gene sequence of Lachnoanaerobaculum orale N1T (98.5%) and Lachnoanaerobaculum saburreum CCUG 28089T (97.6%). The major fatty acids of strain ChDC B114T were C16:0 (30.7%), C14:0 (17.7%), iso-C19:0 (14.9%), and C17:0 2OH (12.0%). The draft genome of strain ChDC B114T was 3,097,953 bp in length. The G+C content of the strain was 35.9 mol %. Average nucleotide identity values between strain ChDC B114T and L. orale N1T and L. saburreum CCUG 28089T were 83.2% and 82.0%, respectively. Genome-to-genome distance values between strain ChDC B114T and L. orale N1T and L. saburreum CCUG 28089T were 26.8% (24.5–29.3%) and 26.30% (24.0–28.8%), respectively. Based on these results, strain ChDC B114T (= KCOM 2030T = JCM 33452T) should be classified as a novel species of genus Lachnoanaerobaculum, for which the name Lachnoanaerobaculum gingivalis sp. nov. is proposed.

Isolation, Characterisation and Complete Genome Sequence of a Tequatrovirus Phage, Escherichia phage KIT03, Which Simultaneously Infects Escherichia coli O157:H7 and Salmonella enterica Abstract Escherichia coli O157:H7 and Salmonella enterica subsp. enterica are the pathogens that frequently cause foodborne illness. Bacteriophage applications have been proposed as effective for preventing food contamination caused by these pathogenic bacteria. Escherichia phage KIT03 was isolated from the soil of a poultry farm in Kyoto, Japan. KIT03 can infect Escherichia coli O157:H7 and Salmonella enterica serotypes Choleraesuis and Enteritidis. One-step growth analysis revealed that KIT03 can propagate within its initial host (E. coli NBRC 3972), E. coli O157:H7 and S. Choleraesuis with an approximate burst size of 39, 51 and 37 phage particles per infected cell, respectively. The morphological type and genome annotation suggested that KIT03 belongs to the family Myoviridae, subfamily Tevenvirinae, genus Tequatrovirus. In vitro challenge tests demonstrated that KIT03 can lyse the tested bacteria and suppress their growth. Based on the susceptibility test and adsorption assay of KIT03 with E. coli K-12 BW25113 mutants, it was proposed that KIT03 may recognise and infect bacteria with a deficient outer core of lipopolysaccharides.

Isolation and Characterization of a New Ferulic-Acid-Biotransforming Bacillus megaterium from Maize Alkaline Wastewater (Nejayote) Abstract Nejayote is an alkaline wastewater generated during the nixtamalization process. Nejayote contains high-value compounds such as ferulic acid (FA), which is widely employed as a substrate for the biotechnological production of flavors and aromas. In the present study, the isolation, identification, and characterization of a native strain of Bacillus megaterium were performed, and its capacity to produce 4-vinylguaiacol (4VG) from ferulic acid was evaluated by employing growing cell and resting cell systems. Growing cells of native B. megaterium biotransformed 6 mM crude FA in nejayote into 2.1 mM 4VG, reaching a productivity of 0.21 mM h−1 4VG, while nejayote enriched with FA at 10, 15, and 25 mM resulted in the formation of 2.4, 3.8, and 6.2 mM 4VG and productivities of 0.24, 0.38, and 0.51 mM h−1 4VG, respectively. In the resting cell system, from 6 and 25 mM pure FA, 3.5 mM 4VG was produced (0.18 mM h−1 4VG), while at 10 and 15 mM FA, 4.6 and 5.1 mM 4VG (average of 0.24 mM h−1 4VG) were obtained, respectively. The native B. megaterium strain, isolated from nejayote, showed great biotechnological potential to produce 4VG from crude FA contained in this wastewater, in which other Bacillus species, such as B. licheniformis and B. cereus, were unable to grow and biotransform FA into 4VG.

Study of the Structure and Biological Activity of the Amino-Terminus of the α-Toxin from Clostridium welchii Type A Abstract To explore the biological activity of Clostridium welchii α-toxin (CPA), the Asp56 residue of CPA was mutated to glycine (CPA D56G) by site-directed mutagenesis, and the 250 amino acid amino-terminal phospholipase C (PLC)-containing domain of CPA (PLC1-250) was isolated. The secondary and three-dimensional (3D) structures of CPA D56G and PLC1-250 were predicted, and the results showed that the secondary structures of CPA D56G and PLC1-250 were composed of α-helices and random coils. The 3D structures of CPA D56G and PLC1-250 were similar to the 3D structures of CPA. The circular dichroism (CD) spectrum of CPA D56G differed from the CD spectrum of CPA, but the CD spectrum of PLC1-250 was similar to the CD spectrum of CPA. Biological activity assays showed that CPA D56G lost the PLC activity of CPA and that mice immunized with CPA D56G were protected against a challenge with 1 MLD C. welchii type A strain C57-1. In addition, PLC1-250 contained the PLC activity of CPA. This study laid a solid foundation for future studies on the relationship between the molecular structure and biological function of CPA and its molecular mechanism. Our study also provided CPA D56G as a candidate strain for engineering a CPA subunit vaccine for C. welchiitype A.

Pseudomonas ovata sp. nov., Isolated from the Skin of the Tail of Farmed Murray cod ( Maccullochella peelii peelii ) with a Profound Ulceration Abstract A Gram-negative, strictly aerobic, motile, rod-shaped bacterium with monopolar flagellum, designated as F51T, was isolated from the skin ulcer of farmed Murray cod sampled from Zhejiang Province, China. Strain F51T grew at 4–37 °C (optimal temperature, 28 °C), pH 5.0–8.5 (optimal pH, 7.5) and NaCl concentration of 0–6.0% (w/v) (optimal concentration, 2.0%). Phylogenetic analysis based on average nucleotide identity (76.2–78.4%) and in silico DNA–DNA hybridization (22.3–23.2%) values revealed that strain F51T forms a distinct lineage in the clade of genus Pseudomonas with less than 98.9% 16S rRNA gene sequence similarity to type strains of the genus and represents a novel species related most closely to Pseudomonas floridensis LMG 30013T. Three housekeeping genes (rpoB, rpoD and gyrB) of strain F51T were analysed to further confirm that the isolate is distinctly delineated from related Pseudomonas species. Chemotaxonomic analysis indicated that the sole respiratory quinone of strain F51T is Q-9; its predominant cellular fatty acids are C16:0, summed feature 3 (iso-C15:0 2-OH and/or C16:1ω7c), summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C10:0 3-OH; and its major polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified glycolipids, three unidentified phospholipids and an unidentified aminophosphoglycolipid. This composition is typical of the chemotaxonomic attributes of Pseudomonas. Based on its phenotypic, chemotaxonomic and phylogenetic features, strain F51T is considered to represent a novel species for which the name Pseudomonas ovata sp. nov. is proposed. The type strain is F51T (= KCTC 62133T = MCCC 1K03458T).