Uptake of N , N ′-Diacetylchitobiose [(GlcNAc) ₂ ] via the Phosphotransferase System Is Essential for Chitinase Production by Serratia marcescens 2170

Abstract

The chiR gene of Serratia marcescens 2170, encoding a LysR-type transcriptional activator, was identified previously as an essential factor for expression of chitinases and a chitin-binding protein, CBP21. To identify other genes that are essential for chitinase production, transposon mutagenesis with mini-Tn5Km1 was carried out, and 25 mutants that were unable to produce chitinases and CBP21 were obtained. Analysis of the mutated gene of one of the mutants, N22, revealed the presence of a pts operon in this bacterium, and a mutation was found in ptsI in the operon. In addition to its inability to produce chitinase, N22 did not grow well on N-acetyl-D-glucosamine (GlcNAc), (GlcNAc) 2 , and some other carbon sources, most of which were phosphotransferase system (PTS) sugars. Thus, the inability to produce chitinase was assumed to be caused by the defect in uptake of (GlcNAc) 2 via the PTS, considering that (GlcNAc) 2 is the minimal substrate for chitinase induction and the major product of chitin hydrolysis by chitinases of this bacterium. To confirm this assumption, the chb operon, encoding the (GlcNAc) 2 -specific enzyme II permease, was cloned by reference to its Escherichia coli counterpart, and the Serratia chb operon was shown to comprise chbB, chbC, bglA, chbR, and chbG. Disruption of chbC drastically reduced production of chitinases and CBP21 and impaired growth on colloidal chitin. These results indicate that uptake of (GlcNAc) 2 is mediated by the PTS and that the (GlcNAc) 2 -specific enzyme II permease constitutes its major pathway. Since (GlcNAc) 2 uptake is essential for induction of chitinases and CBP21 production, (GlcNAc) 2 appears to be the key molecule in recognition and utilization of chitin by S. marcescens.

Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

“…This signature gene is a conserved member of nag operons and respective regulons in Enterobacteriales and Vibrionales and in some ␣-and ␤-proteobacteria (Table 1 and Table S1). Another committed PTS system for transport of chitobiose in E. coli and other enterobacteria is a part of the chb operon controlled by NagC and an additional regulator ChbR (16,45).…”

Regulation of Chitinase Production by the 5'-Untranslated Region of theybfMinSerratia marcescens2170

“…First, mini-Tn5 Sm/ Sp-lacZ was delivered to wild-type S. marcescens 2170 via conjugation with E. coli S17-1(pir) carrying pUTmini-Tn5 Sm/Sp-lacZ. 14) Spectinomycin-resistant transconjugants were obtained using LB plates containing spectinomycin and tetracycline, and these were transferred to master plates. Transposon mutants were grown for 48 h on colloidal chitin agar plates containing spectinomycin.…”

“…Transposon mutagenesis was conducted as described previously. 14) Mutants with altered chitobiase activity when grown on a colloidal chitin plate were detected by means of 4-MU-GlcNAc hydrolysis and isolated. Four of approximately 6,000 transposon mutants, TT116, TT120, TT401, and TT327, were selected based on their chitobiase-deficient phenotype.…”

“…15) In addition, chbC, a (GlcNAc) 2 -specific enzyme IIC component of the PTS, is essential for the induction of chitobiase. 14) Therefore, y To whom correspondence should be addressed. Tel: +81-25-262-6646; Fax: +81-25-262-6854; E-mail: ksuzuki@agr.niigata-u.ac.jp Abbreviations: GlcNAc, N-acetylglucosamine; (GlcNAc) 2 , N,N 0 -diacetylchitobiose; 5 0 -UTR, 5 0 -untranslated region; RT-PCR, reverse transcription polymerase chain reaction; RACE, rapid amplification of cDNA ends uptake of (GlcNAc) 2 is important for the induction of chitobiase as well as that of chitinases and CBP21.…”

“…Disruption of the (GlcNAc) 2 -specific enzyme II permease, encoded by the chb operon, drastically reduced the induction of chitinases and CBP21. 14) S. marcescens 2170 also produces a 98.5-kDa chitobiase, which is mainly located in the periplasm, and hydrolyzes the -1,4-glycosidic bond of (GlcNAc) 2 .…”