This paper deals with the optimization of culture conditions for the production of cholesterol oxidase (COD) by Streptomyces lavendulae NCIM 2499 using the one-factor-at-a-time method, orthogonal array method and response surface methodology (RSM) approaches. The one-factor-at-a-time method was adopted to investigate the effects of medium components (i.e. carbon and nitrogen) and environmental factors (i.e. initial pH) on biomass growth and COD production. Subsequently, an L12 orthogonal matrix was used to evaluate the significance of glycerol, soyabean meal, malt extract, K2HPO4, MgSO4 and NaCl. The effects of media components were ranked according to their effects on the production of COD as malt extract > soyabean meal > K2HPO4 > NaCl > MgSO4 > glycerol. The subsequent optimization of the four most significant factors viz. malt extract, soyabean meal, K2HPO4 and NaCl, was carried out by employing a central composite rotatable design (CCRD) of RSM. There was a 2.48-fold increase in productivity of COD as compared to the unoptimized media by using these statistical approaches.
Fermented milk is a very delicious and nutritional food distributed in the international markets. Rapid preliminary identification of lactic acid bacteria to the species level is an important issue for the fermentation industry. One hundred seventy-one strains of lactic acid bacteria (LAB) were isolated from twenty-nine kurut samples and fifteen traditional fermented mongolian cattle milk (FMCM) samples in Tibet, China. All of the strains were isolated and divided into bacilli or cocci by phenotype, and then differentiated by restriction fragment length polymorphism (RFLP) analysis using a set of restriction enzymes, AluI, HaeIII, BsmaI, TspRI and HinfI. Restriction pattern analyses indicated that the lactobacilli could be clearly identified at the species level and the LAB cocci at the genera level by the five restriction enzymes. Further studies on 16S rDNA sequences of representative and undefined strains showed that the combination of RFLP analysis of polymerase chain reaction (PCR) for amplifying 16S rDNA and 16S rDNA sequence analysis is rapid, easy to perform, and effective for large-scale preliminary identification of LAB.
Edwardsiella tarda is one of the leading marine pathogens that can infect a wide range of cultured marine species. In this study, the acrR-acrAB cluster was cloned from TX1, a pathogenic E. tarda strain isolated from diseased fish. AcrR and AcrAB were found to be involved in resistance against acriflavine and methyl viologen, which positively regulate the expression of acrAB. AcrR negatively regulates its own expression and the expression of the acrAB operon, most likely by interacting with a 24-bp operator site that overlaps the putative promoter of acrA (PacrA). The repressive effect of AcrR on PacrA could be relieved by acriflavine, methyl viologen, and ethidium bromide, the presence of each of which enhanced transcription from PacrA. Interruption of the regulated expression of acrR by introducing into TX1 a plasmid that overexpresses acrR affected growth under stress conditions, AI-2 production, and bacterial virulence. In addition, mutational analyses identified a constitutively active AcrR mutant (named N215), which exhibits full repressor activity but is impaired in its ability to interact with the inducer. Overexpression of N215 produced the same kind of but moderately stronger effect on TX1 compared to that produced by overexpression of the wild-type acrR.
Bacterial strain YM22-133T was isolated from the sediment of an estuary and grew in media with an artificial seawater base. Strain YM22-133T was Gram-positive, aerobic, non-motile and rod shaped. The cell-wall peptidoglycan contained LL-DAP, glycine, alanine and hydroxyglutamate. The predominant menaquinone was MK-9 (H8), with MK-9 (H0), MK-9 (H2), MK-9 (H4) and MK-9 (H6) present as minor menaquinones. The G+C content of the genomic DNA from the strain was 68 mol%. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain is nearest to Acidimicrobium ferrooxidans DSM 10331T. However, the similarity is relatively low (87.1%) and the physiological characteristics are also different: Acidimicrobium ferrooxidans is thermotolerant and acidophilic. Therefore, strain YM22-133T can be classified as a novel genus and species, Ilumatobacter fluminis gen. nov., sp. nov. (type strain YM22-133T =DSM 18936T=MBIC08263T).
This study reports phenotypic and molecular characterization of a novel CTX-M β-lactamase carried by two Klebsiella pneumoniae isolates collected from two hospitals in China. Conjugation experiment, Southern hybridization, susceptibility testing, isoelectric focusing, PCR, and sequencing techniques as well as clone, expression, purification and kinetics were carried out to describe the characterization of the novel CTX-M-type enzyme. The analyses of plasmid profiling and pulsed-field gel electrophoresis of the novel enzyme were performed to investigate epidemiology. The PCR products had 967 nucleotides and a novel CTX-M enzyme with a pI of 8.5 was implicated in this resistance: CTX-M-72. Two strains exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. The amino acid sequence of the CTX-M-72 β-lactamase differed from that of the CTX-M-3 β-lactamase by the Arg→Gly change at position 164. The novel enzyme was susceptible to ceftazidime, the same response being observed for other CTX-M enzymes. The substrates of the β-lactamase were also characterized. Furthermore, two resistant genes of clinical strains were closely related. The emergence of a novel CTX-M-type extended-spectrum β-lactamase was rarely described in other areas. This study illustrated the importance of molecular surveillance in tracking CTX-M-producing strains in large teaching hospitals, suggested the horizontal transfer of plasmid-borne blaCTX-M genes contributed to the dissemination of CTX-M enzymes in hospital environments, and emphasized the need for epidemiological monitoring.
A Gram-variable, spore-forming, motile rod, designated strain Y1T, was isolated from the hopper surface of equipment used for soy sauce production. Phylogenetic analysis based on 16S rRNA gene sequence revealed that Y1T is affiliated phylogenetically to the genus Sporosarcina, and the strain showed sequence similarities of 95.8-99.2% to those of Sporosarcina species with validly published names. The values of DNA-DNA relatedness between strain Y1T and related type strains of the genus Sporosarcina were below 27%. The major cellular fatty acids were iso-C15:0 and anteiso-C15:0. The cell-wall peptidoglycan was of the A4α type (Lys-Glu) and the major isoprenoid quinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G+C content of the strain was 43.6 mol%. On the basis of phylogenetic analysis and physiological and chemotaxonomic data, the isolate represents a novel species of the genus Sporosarcina, for which the name Sporosarcina luteola sp. nov. is proposed. The type strain is strain Y1T (=JCM 15791T=NRRL B-59180T=NBRC 105378T=CIP 109917T=NCIMB 14541T).
A Gram-positive, spore-forming, motile rod-shaped bacterium, designated strain Y27T, was isolated from the bottom of a mold fermenter used in the process of soy sauce production. Phylogenetic analysis of the 16S rRNA gene sequence from this strain placed it within the genus Oceanobacillus, and further sequence analysis revealed that this strain has a sequence similarity of 95.0-98.7% to other known species of Oceanobacillus. The DNA-DNA relatedness between strain Y27T and related type strains of the genus Oceanobacillus is below 43%, indicating that it should be considered a separate species. Characterization of strain Y27T revealed that the major cellular fatty acid is anteiso-C15:0, the cell wall contains meso-diaminopimelic acid-type peptidoglycans, the major menaquinone is MK-7, and the major polar lipids are diphosphatidylglycerol and phosphatidylglycerol. The genomic DNA G+C content of the strain is 38.0 mol%. On the basis of these phylogenetic, physiological and chemotaxonomic data, we propose that this isolate represents a novel species of the genus Oceanobacillus, and propose the name Oceanobacillus soja sp. nov. The type strain is strain Y27T (=JCM 15792T=NRRL B-59181T=NBRC 105379T=NCIMB 14542T).