In our recent work, we found that pyrrolnitrin, and not phenazines, contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in the regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in the regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.
Susceptibility testing of bacteria to disinfecting chemical agents isolated from dental unit waterlines (DUWL) is necessary for the development of effective disinfectant products. However, until now, susceptibility tests for chemical agents, which are components of DUWL disinfectant products, have not been conducted on bacteria isolated from DUWL water. The aim of this study was to evaluate and compare the susceptibilities of DUWL isolates in planktonic and biofilm states to cetylpyridinium chloride, as well as to the four chemical agents currently used for DUWL management. A total of 56 isolates, including 12 genera, were identified by 16S rDNA sequencing, and one strain of each genus was selected for susceptibility testing. A total of 12 isolates were used for the susceptibility tests. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the planktonic state and the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) for the biofilm state using microtiter plates. MIC, MBC, MBIC, and MBEC of the 12 isolates for ethanol were the highest, followed by sodium hypochlorite, hydrogen peroxide, and chlorhexidine. Similar to chlorhexidine, the lowest MIC, MBC, MBIC, and MBEC were found in cetylpyridinium chloride. The susceptibilities of the isolates for sodium hypochlorite and ethanol were similar in the planktonic and biofilm states. For hydrogen peroxide and chlorhexidine, the MBIC and MIC were similar, but MBEC was 256 times higher than MBC. The MBIC and MBEC of isolates for cetylpyridinium chloride were 128 and 256 times higher than the MIC and MBC, respectively. As far as we know, this was the first study reporting the susceptibility of DUWL isolates to cetylpyridinium chloride and chemical agents used for disinfecting DUWLs. Cetylpyridinium chloride, for which the DUWL isolates showed the highest susceptibility, could be used for disinfecting DUWLs.
A gene (aga0917) encoding a putative β-agarase was identified from the genome of Pseudoalteromonas fuliginea YTW1-15-1. The nucleotide sequence analysis revealed that aga0917 had significant homology to the agarase genes of the GH16 family. aga0917 encodes a putative protein of 290 amino acids with an estimated molecular mass of 32.5 kDa, including a 21-amino acid signal peptide. A gene fragment encoding only the putative mature form of Aga0917 (269 amino acids) was overexpressed in Escherichia coli BL21 (DE3) pLysS as a 6 × histine-tagged fusion protein (rmAga0917). The Km, Vmax, and kcat for agarose of rmAga0917 were 39.6 mg/mL, 334 (U/mg) of protein, and 178 (1/s), respectively. According to the results of thin-layer chromatography and mass spectrometry analysis, the main end product from agarose with rmAga0917 was neoagarotetraose, in addition to a small amount of neoagarobiose. Notably, the recombinant protein rmAga0917 showed optimum activity at 60°C and retained approximately 100% agarolytic activity after being kept at 40°C for 1 h and 57% residual activity after incubation at 50°C for 1 h. The rmAga0917 exhibited maximum agarase activity at pH 6.0, and retained more than 80% of activity after incubation over a range of pH 4.0–9.0 for 1 h at 4°C.
The emergence of antibiotic resistance among multidrug-resistant (MDR) microbes is of growing concern, and threatens public health globally. A total of 129 Escherichia coli isolates were recovered from lowland aqueous environments near hospitals and medical service centers in the vicinity of Kuala Lumpur, Malaysia. Among the eleven antibacterial agents tested, the isolates were highly resistant to trimethoprim-sulfamethoxazole (83.7%) and nalidixic acid (71.3%) and moderately resistant to ampicillin and chloramphenicol (66.7%), tetracycline (65.1%), fosfomycin (57.4%), cefotaxime (57.4%), and ciprofloxacin (57.4%), while low resistance levels were found with aminoglycosides (kanamycin, 22.5%; gentamicin, 21.7%). The presence of relevant resistance determinants was evaluated, and the genotypic resistance determinants were as follows: sulfonamides (sulI, sulII, and sulIII), trimethoprim (dfrA1 and dfrA5), quinolones (qnrS), β-lactams (ampC and blaCTX-M), chloramphenicol (cmlA1 and cat2), tetracycline (tetA and tetM), fosfomycin (fosA and fosA3), and aminoglycosides (aphA1 and aacC2). Our data suggest that multidrug-resistant E. coli strains are ubiquitous in the aquatic systems of tropical countries and indicate that hospital wastewater may contribute to this phenomenon.
Fungi are the most suitable cellulase producers attributing to its ability to produce a complete cellulase system. 33 Genus, 175 Species fungi were isolated from Sanya mangrove, Hainan, China. Using congo red cellulose (CMC) medium, five fungi of cellulose-degrading were selected for further study. Molecular biology and morphological identification showed that all of these five fungi belong to Aspergillus fungi. The cellulase produced by these fungi were monitored during liquid state fermentation. The optimum conditions study for enzyme production illustrated that the highest activities appeared at pH 3.0, 35°C after fermentation for 3 days. Beyond that, the enzyme activity of mixed fungi is 11–26% higher than pure. The study demonstrated that mixed culture improved the hydrolysis of fungi cellulase.
We previously showed that nuclear DNA replication (NDR) is regulated by a checkpoint monitoring the occurrence of organelle DNA replication (ODR) in a unicellular red alga Cyanidioschyzon merolae. These analyses depended on the use of chemical CDK inhibitors such as CDK2 inhibitor II and roscovitine, but subsequent analyses yielded conflicting results depending on the experimental conditions. In the present study, we identified significantly short half-lives of the used chemicals in the sulfur acidic cultivation medium, which reconciles the discrepancy among these results.