The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli

We are studying exoproteins of the enteric bacterium Serratia marcescens as a model system for the release of extracellular proteins from the cell. In this work we report the cloning of the gene for a secreted nuclease from S. marcescens and its complete nucleotide sequence. Following expression of the nuclease gene in both S. marcescens and Escherichia coli we were able to demonstrate the presence of the nuclease extracellularly in both organisms. Cell lysis did not occur and there was no concurrent release of cytoplasmic or periplasmic proteins. No accessory genes appeared to be required for extracellular secretion of the nuclease from E. coli. We can conclude that E. coli is capable of secreting certain proteins extracellularly, and may be a suitable host organism for the genetic analysis of extracellular protein secretion when provided with a suitable protein to export.     

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.     

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli.

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.     

Differential secretion of isoforms of Serratia marcescens extracellular nuclease.

Extracellular secretion of the Serratia marcescens nuclease occurs in a two-step process: (i) rapidly to the periplasm via a signal sequence-dependent pathway and then (ii) slowly to the extracellular growth medium without cell lysis. There are two major isoforms of the nuclease in the culture supernatant of S. marcescens. We have isolated, purified, and determined the sequences of both isoforms. The first isoform, the mature nuclease (Sm2), is the result of signal sequence processing. The second isoform (Sm1) has three additional amino acids missing from the N terminus of the mature nuclease. Sm1 starts to appear extracellularly only during prolonged growth of a culture (16 to 48 h), probably because of cell lysis. However, pulse-chase experiments show that it is made early with Sm2 but is not secreted efficiently.     

Multi-copy repression of Serratia marcescens nuclease expression by dinI

The dinI homolog of S. marcescens was cloned from a plasmid library by virtue of its ability to inhibit nuclease expression from the S. marcescens nucA gene integrated in the genome of E. coli. The S. marcescens DinI protein is 68% identical to DinI of E. coli. It has a similar effect on other SOS regulated genes and likely exerts it effect on nuclease expression, which is most pronounced as the cells entered stationary phase, through inhibition of basal SOS expression. Received: 12 April 2001 / Accepted: 14 May 2001     

Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli.

A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with Tn1000 and Tn5 IS50L::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hly, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxy-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (Mr 504) to maltoheptaose (Mr 1,152) and was totally abolished by dextran 4 (Mr 4,000). This result and the observed influx of [14C]sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin.     

Molecular cloning and expression of the Streptococcus lactis tagatose 1,6-bisphosphate aldolase gene in Escherichia coli

Abstract A 4.4 kb Eco RI DNA fragment of the Streptococcus lactis H₁ plasmid pDI1 was cloned into the Escherichia coli plasmid pACYC 184. The recombinant plasmid expressed d -tagatose 1,6-bisphosphate aldolase activity in E. coli . Enzyme activity was at the same level as in the original S. lactis host but was not repressed by glucose.     

Molecular cloning and physical map of bacteriophage PM2 DNA

The entire genome and the DNA fragments of the lipid-containing bacteriophage pM2 were cloned in the pBR322 plasmid vector. A physical map including the sites for the following restriction enzymes was obtained: HpaII, HaeIII, TthI, Sau96I, AvaII, PstI, BstNI, AccI, HincII, HpaI and HindIII. No restriction sites on PM2 DNA were found for BalI, BamHI, BclI, BglI, BglII, BstEII, KpnI, PvuII, SacI, SalI, Sau3A, XbaI and XhoI.     

Molecular cloning of an ice nucleation gene from Erwinia ananas and its expression in Escherichia coli

Abstract An approximately 7 kbp genomic DNA fragment was cloned from an ice nucleation-active (ina) strain of Erwinia ananas and defined as to its restriction enzyme site. When the DNA fragment was introduced into E. coli MM294, a potent ice nucleation activity was expressed. Both 0.7 kbp truncation from the 5'-end and 1.7 kbp truncation from the 3'-end were also effective in expressing the ice nucleation activity in E. coli . Therefore, the resulting DNA fragment of approximately 5 kbp was considered to be an ina gene and named ina A. It existed as a unique gene in this strain of E. ananas . No corresponding ina gene existed in an ice nucleation-inactive strain of E. milletiae .     

Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis

Prodigiosin, the bright red pigment produced by many strains of Serratia marcescens, is synthesized by a bifurcated pathway that terminates in the enzymatic condensation of the two final products, a monopyrrole and a bipyrrole . Sau3A fragments of S. marcescens ( Nima ) DNA were introduced into a strain of Escherichia coli K-12 by use of the cosmid vector pHC79 , and transformed clones were selected based on resistance to ampicillin. Among 879 transformants screened, 2 could be induced to synthesize prodigiosin when supplied with either one or both terminal products of the bifurcated pathway. Data are presented to support the idea that production of prodigiosin is not usually mediated by a plasmid.     

Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima)

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C.     

Overproduction of the Bacillus sphaericus R modification methylase in Escherichia coli and its purification to homogeneity

A DNA fragment containing the information coding for the GGCC-specific Bacillus sphaericus R modification methylase, BspR, was inserted into plasmid vector pKK223-3 under the control of the strong and inducible tac promoter, and transformed into Escherichia coli HB101. Upon induction this strain accumulated the methylase enzyme (while cell growth was inhibited) up to several percent of total cellular protein. Homogeneous methylase could be prepared in three purification steps.     

Action of hexaamminecobalt on the activity of Serratia marcescens nuclease

Using CD spectroscopic and kinetic analysis, a refined mechanism of Co(NH₃) ₆ ³⁺ action on activity of Serratia marcescens nuclease was elucidated. The mechanism was identical with previously found mechanisms of Mg²⁺ and C₇H₅O₂Hg⁺. Similarly to Mg²⁺ and C₇H₅O₂Hg⁺, Co(NH₃) ₆ ³⁺ binding to the DNA substrate induced changes in the secondary structure which resulted in changes of the enzymatic activity of the S. marcescens nuclease. Upon binding of 0.03 Co(NH₃) ₆ ³⁺ per DNA phosphate, highly polymerized DNA displayed A-form characteristics. The DNA transition from B-form to A-form intermediate was followed by a decrease of the nuclease activity. The diminishing nuclease activity was consistent with diminishing values of Km and Kcat. Co(NH₃)₆ ³⁺ binding to the highly polymerized DNA caused a 1.7–2.8-fold decrease in Km, and 13.3–19.9 decrease in Vmax compared with Mg-DNA complex. A vast excess of Co(NH₃)₆ ³⁺ did not affect the activity of S. marcescens nuclease if the DNA in the assay mixture remained in its B-form conformation. Preincubation of S. marcescens nuclease with Co(NH₃)₆ ³⁺ did not influence the tertiary structure of the enzyme.     

Molecular cloning of the fdhF gene of Escherichia coli K-12

Abstract The fdhF gene of Escherichia coli , coding for at least one component of benzyl viologen-linked formate dehydrogenase (FDH-BV) activity, was isolated on a ColE1- fdhF hybrid plasmid from the Clarke and Carbon colony bank.
Endonuclease restriction maps of this plasmid and its pBR322-subcloned derivative, pLW06, were constructed. Various hybrid plasmids were further obtained by deletion of endonuclease-cleaved fragments from pLW06 DNA. Their complementation pattern was analyzed after introduction into different fdhF mutant strains. The fdhF gene was shown to be located on a 5.5 kb Bam HI- Pvu II-DNA fragment, which restored FDH-BV activity to the wild-type level.