The effect of nuclease on transformation efficiency in Serratia marcescens

Palomar, J; Guasch, Joan Francesc; Regué, Miguel; Viñas, Marc

doi:10.1016/0378-1097(90)90076-3

Cited by 7 publications

(6 citation statements)

References 22 publications

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“…S. marcescens 2170 (25) was used for chitinase production and Tn5 insertion mutagenesis and as a source of chromosomal DNA for chitinase gene cloning. Chitinases produced by S. marcescens 2170 were compared with those of S. marcescens QMB1466 (24) obtained from the American Type Culture Collection.…”

Section: Methodsmentioning

confidence: 99%

“…Tn5 transposon mutagenesis of S. marcescens 2170 harboring the plasmid pTROY11 was carried out as described previously (25). Kanamycin-resistant colonies were transferred onto agar plates of YEM medium containing 0.2% colloidal chitin, 50 g of kanamycin per ml, and 2 mg of ampicillin per ml and were incubated at 30°C.…”

Section: Methodsmentioning

confidence: 99%

“…However, this bacterium is not a suitable model for the genetic analysis of the degradation and utilization of chitin. Therefore, we chose to study Serratia marcescens 2170, an active producer of chitinase (9,25,27) and an excellent model for studying the degradation and utilization of chitin. S. marcescens is an efficient degrader of chitin, and the chiA and chiB genes encoding chitinases A and B of three S. marcescens strains, QMB1466, BJL200, and 27117, have been cloned and sequenced (5, 6, 10-12, 16, 17).…”

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Genetic analysis of the chitinase system of Serratia marcescens 2170

et al. 1997

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To carry out a genetic analysis of the degradation and utilization of chitin by Serratia marcescens 2170, various Tn5 insertion mutants with characteristic defects in chitinase production were isolated and partially characterized. Prior to the isolation of the mutants, proteins secreted into culture medium in the presence of chitin were analyzed. Four chitinases, A, B, C1, and C2, among other proteins, were detected in the culture supernatant of S. marcescens 2170. All four chitinases and a 21-kDa protein (CBP21) lacking chitinase activity showed chitin binding activity. Cloning and sequencing analysis of the genes encoding chitinases A and B of strain 2170 revealed extensive similarities to those of other strains of S. marcescens described previously. Tn5 insertion mutagenesis of strain 2170 was carried out, and mutants which formed altered clearing zones of colloidal chitin were selected. The obtained mutants were divided into five classes as follows: mutants with (i) no clearing zones, (ii) fuzzy clearing zones, (iii) large clearing zones, (iv) delayed clearing zones, and (v) small clearing zones. Preliminary characterization suggested that some of these mutants have defects in chitinase excretion, a negatively regulating mechanism of chitinase gene expression, an essential factor for chitinase gene expression, and a structural gene for a particular chitinase. These mutants could allow researchers to identify the genes involved in the degradation and utilization of chitin by S. marcescens 2170.The number of studies dealing with bacterial chitinasestheir biochemical properties, the structure of the genes encoding them, the catalytic mechanism involved, and their tertiary structures-has been increasing rapidly. The hydrolysis of chitin by chitinases is the most critical step in the degradation and utilization of chitin by bacteria. However, the study of chitinases is not sufficient to elucidate the process by which chitin is degraded and utilized by bacteria. The process involves a number of steps, including the recognition of chitin outside of the cell, the induction of chitinases, the maintenance of proper levels of chitinase production, and the incorporation and catabolism of degradation products. In this study our intent was to identify the genes involved in the degradation and utilization of chitin. Our long-term goal is to answer the following questions. How do bacteria recognize chitin? How is chitinase production induced and regulated? Why do chitinolytic bacteria produce multiple chitinases? How are degradation products processed? Our ultimate goal in these studies is a general understanding of how bacterial cells degrade and utilize chitin.We have studied the chitinase system of Bacillus circulans WL-12 and have provided comprehensive findings on biochemical properties, structure-function relationships, the identification of essential amino acid residues for catalytic activity, and the mechanisms by which multiple chitinases are generated (1, 2, 4, 23, 31-35). However, this bacterium is not a suitable...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Genetic analysis of the chitinase system of Serratia marcescens 2170

et al. 1997

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“…Isolation of S. marcescens reg::Tn5 mutant. Tn5 transposon mutagenesis of S. marcescens 2170 with lambda 467 phage (::Tn5) was carried out as previously described (37). Kanamycin (25 g/ml)-resistant colonies were screened for lower levels of bacteriocin production upon mitomycin treatment by using the agar overlay test (40).…”

Section: Methodsmentioning

confidence: 99%

Genetic evidence for an activator required for induction of colicin-like bacteriocin 28b production in Serratia marcescens by DNA-damaging agents

et al. 1996

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Bacteriocin 28b production is induced by mitomycin in wild-type Serratia marcescens 2170 but not in Escherichia coli harboring the bacteriocin 28b structural gene (bss). Studies with a bss-lacZ transcriptional fusion showed that mitomycin increased the level of bss gene transcription in S. marcescens but not in the E. coli background. A S. marcescens Tn5 insertion mutant was obtained (S. marcescens 2170 reg::Tn5) whose bacteriocin 28b production and bss gene transcription were not increased by mitomycin treatment. Cloning and DNA sequencing of the mutated region showed that the Tn5 insertion was flanked by an SOS box sequence and three genes that are probably cotranscribed (regA, regB, and regC). These three genes had homology to phage holins, phage lysozymes, and the Ogr transcriptional activator of P2 and related bacteriophages, respectively. Recombinant plasmid containing this wild-type DNA region complemented the reg::Tn5 regulatory mutant. A transcriptional fusion between a 157-bp DNA fragment, containing the apparent SOS box upstream of the regA gene, and the cat gene showed increased chloramphenicol acetyltransferase activity upon mitomycin treatment. Upstream of the bss gene, a sequence similar to the consensus sequence proposed to bind Ogr protein was found, but no sequence similar to an SOS box was detected. Our results suggest that transcriptional induction of bacteriocin 28b upon mitomycin treatment is mediated by the regC gene whose own transcription would be LexA dependent.Serratia marcescens has been shown to produce bacteriocins upon induction with DNA-damaging agents (50). These bacteriocins have been classified into two groups (23): fraction 1 bacteriocins are active against Escherichia coli but not against S. marcescens, and fraction 2 bacteriocins are active against S. marcescens but not against E. coli (50). Bacteriocins belonging to fraction 1 are simple polypeptides that resemble colicins (50). Only colicin-like bacteriocins L and 28b have been studied in some detail. Bacteriocin L from S. marcescens JF246 has been isolated and characterized, and the effects of this bacteriocin on the incorporation of labelled leucine and thymidine and on the cellular levels of ATP in E. coli were similar to those produced by pore-forming colicins (17,18,40). On the other hand, the bacteriocin 28b structural gene (bss) has been cloned and sequenced, and the predicted amino acid sequence of the C-terminal part of this bacteriocin has been shown to have a high degree of similarity to the C-terminal domains of pore-forming colicins (56). The two bacteriocins are very closely related, if not the same, and similar bacteriocins are produced by most S. marcescens biotypes (21).Colicin production is induced by mitomycin and other DNAdamaging agents (38). Determinants for colicin production studied so far are encoded by either small high-copy-number colicinogenic plasmids (type I) or large low-copy-number colicinogenic plasmids (type II) (38). An important feature of these plasmids is that they confer on their host...

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“…All S. marcescens 2170 (Palomar et al, 1990) and E. coli K-12 strains and plasmids used in the present study are listed in Table 1. Luria-Bertani (LB) growth medium (Miller, 1972) was used for routine cultures and biofilm formation assays.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Csr system in Serratia marcescens 2170

Ito

Nomura

Sugimoto

et al. 2014

J. Gen. Appl. Microbiol.

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The carbon storage regulator (Csr) global regulatory system is conserved in many eubacteria and coordinates the expression of various genes that facilitate adaptation during the major physiological growth phase. The Csr system in Escherichia coli comprises an RNA-binding protein, CsrA; small non-coding RNAs, CsrB and CsrC; and a decay factor for small RNAs, CsrD. In this study, we identified the Csr system in Serratia marcescens 2170. S. marcescens CsrA was 97% identical to E. coli CsrA. CsrB and CsrC RNAs had typical stem-loop structures, including a GGA motif that is the CsrA binding site. CsrD was composed of N-terminal two times transmembrane region and HAMP-like, GGDEF, and EAL domains. Overexpression of S. marcescens csr genes complemented the phenotype of E. coli csr mutants. S. marcescens CsrD affected the decay of CsrB and CsrC RNAs in E. coli. These results suggest that the Csr system in S. marcescens is composed of an RNA-binding protein, two Csr small RNAs, and a decay factor for Csr small RNAs.Key words: Csr system; EAL domain; GGDEF domain; Serratia marcescens; small RNA IntroductionThe carbon storage regulator (Csr) and homologous repressor of secondary metabolite (Rsm) systems, which are the global regulatory systems found in many eubacteria, coordinate the expression of various genes that facilitate adaptation during the major physiological growth phase (Romeo, 1998). In Escherichia coli, the Csr system is composed of a 61-amino-acid RNA-binding protein, CsrA; 366 and 245 nucleotide (nt) small non-coding RNA (sRNA) molecules, CsrB and CsrC, respectively (Liu and Romeo, 1997;Weilbacher et al., 2003); and a predicted membranebound signaling protein, CsrD . CsrA functions as a dimer that binds to specific mRNAs, resulting in altered translation and/or message stability Mercante et al., 2006Mercante et al., , 2009Schubert et al., 2007). CsrA represses stationary-phase gene expression and processes, including gluconeogenesis, glycogen biosynthesis, glycogen catabolism, and biofilm formation, but activates glycolysis, acetate metabolism, and motility (Babitzke and Romeo, 2007;Romeo, 1998). The mechanism underlying CsrA-mediated repression of glycogen biosynthesis (glgCAP) has been elucidated in detail (Baker et al., 2002;Mercante et al., 2009). CsrA recognizes and specifically binds to the untranslated leader of glgCAP mRNA. One of the four binding sites overlaps the glgC Shine-Dalgarno (SD) sequence. The binding of CsrA to these sites regulates GlgC synthesis by preventing ribosome binding, leading to rapid degradation of the transcript (Baker et al., 2002;Mercante et al., 2009;Romeo, 1998).CsrA also activates gene expression, and an activation mechanism has been reported recently. The expression of flhDC, which encodes the master regulator of flagella synthesis and chemotaxis, was activated by CsrA (Wei et al., 2001). CsrA binds to two sites in the flhDC leader transcript and stabilizes it by inhibiting the 5 -end-dependent RNase E cleavage pathway (Yakhnin et al., 2013). A GGA motif is a highly con...

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The effect of nuclease on transformation efficiency in Serratia marcescens

Cited by 7 publications

References 22 publications

Genetic analysis of the chitinase system of Serratia marcescens 2170

Genetic analysis of the chitinase system of Serratia marcescens 2170

Genetic evidence for an activator required for induction of colicin-like bacteriocin 28b production in Serratia marcescens by DNA-damaging agents

Identification of a Csr system in Serratia marcescens 2170

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