Prombutara P, Allen M S
Department of Biological Sciences, University of North Texas, Denton, Texas, USA
Department of Biological Sciences, University of North Texas, Denton, Texas, USA Center for Biosafety and Biosecurity, University of North Texas Health Science Center, Fort Worth, Texas, USA Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, Texas, USA.
Appl Environ Microbiol. 2015 Dec 28;82(6):1646-52. doi: 10.1128/AEM.02917-15.
Thauera aminoaromatica MZ1T, a floc-forming bacterium isolated from an industrial activated-sludge wastewater treatment plant, overproduces exopolysaccharide (EPS), leading to viscous bulking. This phenomenon results in poor sludge settling and dewatering during the clarification process. To identify genes responsible for bacterial flocculation, a whole-genome phenotypic-sequencing technique was applied. Genomic DNA of MZ1T flocculation-deficient mutants was subjected to massively parallel sequencing. The resultant high-quality reads were assembled and compared to the reference genome of the wild type (WT). We identified nine nonsynonymous mutations and one nonsense mutation putatively involved in EPS biosynthesis. Complementation of the nonsense mutation located in an EPS deacetylase gene restored the flocculating phenotype. The Fourier transform infrared (FTIR) spectra of EPS isolated from the wild type showed a reduced C=O peak of the N-acetyl group at 1,665 cm(-1) compared to the spectra of MZ1T floc-deficient mutant EPS, suggesting that the WT EPS was partially deacetylated. Gene expression analysis also demonstrated that the putative deacetylase gene transcript increased before flocculation occurred. These data suggest that targeting deacetylation processes via direct chemical modification of EPS or enzyme inhibition may prove useful in combating viscous bulking in this and related bacteria.
氨基芳香索氏菌MZ1T是从一家工业活性污泥废水处理厂分离出的一种形成絮体的细菌，它会过量产生胞外多糖（EPS），导致粘性膨胀。这种现象会导致在澄清过程中污泥沉降和脱水效果不佳。为了鉴定负责细菌絮凝的基因，应用了全基因组表型测序技术。对MZ1T絮凝缺陷型突变体的基因组DNA进行大规模平行测序。将得到的高质量读数进行组装，并与野生型（WT）的参考基因组进行比较。我们鉴定出九个可能参与EPS生物合成的非同义突变和一个无义突变。位于EPS脱乙酰酶基因中的无义突变的互补恢复了絮凝表型。与MZ1T絮凝缺陷型突变体EPS的光谱相比，从野生型分离出的EPS的傅里叶变换红外（FTIR）光谱在1665 cm-1处显示N-乙酰基的C=O峰降低，这表明野生型EPS被部分脱乙酰化。基因表达分析还表明，假定的脱乙酰酶基因转录本在絮凝发生前增加。这些数据表明，通过对EPS进行直接化学修饰或酶抑制来靶向脱乙酰化过程，可能被证明对对抗这种及相关细菌中的粘性膨胀有用。
