Cellulose as an extracellular matrix component present in Enterobacter sakazakii biofilms.

Cellulose was identified and characterized as an extracellular matrix component present in the biofilm of an Enterobacter sakazakii clinical isolate grown in nutrient-deficient (M9) medium. Using a bacterial artificial cloning approach in Escherichia coli and subsequent screening of transformants for fluorescence on calcofluor plates, nine genes organized in two operons were identified as putatively responsible for the biosynthesis of cellulose. In addition to the genes already described for cellulose production, two more genes were identified, putatively transcribed together with the genes from the first operon. Putative cellulose in E. sakazakii ES5 biofilm grown on glass coverslips was visualized by calcofluor staining and confocal fluorescence laser scanning microscopy. For the first time, the presence of cellulose in biofilms produced by E. sakazakii was confirmed by methylation analysis.

Cloning and characterization of Enterobacter sakazakii pigment genes and in situ spectroscopic analysis of the pigment.

Enterobacter sakazakii is considered an opportunistic foodborne pathogen that is characterized by formation of yellow-pigmented colonies. Because of the lack of basic knowledge about Enterobacter sakazakii genetics, the BAC approach and the heterologous expression of the pigment in Escherichia coli were used to elucidate the molecular structure of the genes responsible for pigment production in Enterobacter sakazakii strain ES5. Sequencing and annotation of a 33.025 bp fragment revealed seven ORFs that could be assigned to the carotenoid biosynthesis pathway. The gene cluster had the organization crtE-idi-XYIBZ, with the crtE-idi-XYIB genes putatively transcribed as an operon and the crtZ gene transcribed in the opposite orientation. The carotenogenic nature of the pigment of Enterobacter sakazakii wt was ascertained by in situ analysis using visible microspectroscopy and resonance Raman microspectroscopy.