RcsA is an unstable positive regulator required for the synthesis of colanic acid capsular polysa... more RcsA is an unstable positive regulator required for the synthesis of colanic acid capsular polysaccharide in Escherichia coli. Degradation of the RcsA protein in vivo depends on the ATP-dependent Lon protease. DNA sequence analysis of the rcsA gene reveals a single open reading frame for a 23,500-Da highly basic protein (pl = 9.9), consistent with the observed size of the purified subunit of RcsA. The DNA and protein sequences are highly homologous to the rcsA gene and protein from Kkebsiella pneumoniae and other species. The carboxy-terminal region of RcsA contains a possible helix-turn-helix DNA-binding motif that resembles sequences found at the carboxy terminus of RcsB, another positive regulator of capsule synthesis, and in several other transcriptional regulators including members of the LuxR family. rcsA62, a mutation in rcsA that leads to increased capsule synthesis, encodes a protein designated RcsA*, which differs from wild-type RcsA only in the replacement of Met-145 by valine. The RcsA* protein is subject to Lon-dependent degradation. The stability of wild-type RcsA in vivo is increased by multicopy RcsB. Conversely, RcsA is degraded more rapidly in rcsB mutant hosts than in wild-type hosts. These results suggest that RcsA and RcsB interact in vivo and are consistent with genetic experiments that indicate an interaction between RcsA and RcsB. Based on these experiments, we propose a model for capsule regulation in which RcsA interacts directly with RcsB to promote transcription of the genes for capsule synthesis. Colanic acid capsular polysaccharide synthesis in Esche- richia coli is encoded by the cps genes (51) and is under the control of two positive regulators, the products of the rcsA and rcsB genes (21). RcsA has been identified as an unstable protein, which is normally limiting for capsule synthesis (49). Mutations in the ATP-dependent protease Lon result in increased capsule synthesis; we have previously demon- strated that RcsA is stabilized in lon mutant cells (49). Therefore, it seems likely that the increase in capsule synthesis in lon mutants is due at least in part to the increased accumulation of RcsA in these cells. Why is RcsA unstable? One rationale for incorporating an unstable protein into a regulatory network is provided by the example of the cell division inhibitor SulA, another target of Lon-dependent degradation (34). SulA synthesis is tightly regulated by the LexA repressor, and SulA is only made as part of the SOS response to DNA damage (24, 33). When SulA is present in cells, no septa are formed and cells do not divide (25). Thus, SulA overproduction or constitutive syn- thesis of SulA is lethal to growing cells (20, 25, 42). It is essential that a growing cell rid itself of any SulA made during the SOS response. The Lon-mediated degradation of SulA provides a straightforward mechanism for the recovery from this particular emergency response. Many of the parallel issues for the regulatory circuit for capsule synthesis have not yet been addressed. Overproduction of RcsA from a multicopy plasmid is not itself lethal, but
A new phosphorus-containing compound (1) was detected by 31 P NMR spectroscopy in Streptomyces sp... more A new phosphorus-containing compound (1) was detected by 31 P NMR spectroscopy in Streptomyces sp. A50. Compound 1, 1(R)-O-methyl-2-(N-acetyl)glucoseamine-6-O-phosphate-1(R)-2-(N-acetyl)glucosamine, exhibited a pK a value around zero. The compound was found both in the extracellular culture broth and in the cells. While very low concentrations of 1 were found in the culture broth of other species of Streptomyces, its presence in high concentrations was specific to Streptomyces sp. A50. The highly acidic compound was isolated from the broth, and its structure was elucidated by a combination of 1D-, 2Dhomonuclear, and inverse heteronuclear NMR techniques and mass spectroscopy. We have previously shown that Streptomyces sp. A50 exhibits the unusual capacity to convert up to 30% of the inorganic phosphate (Pi) present in the growth medium into polyphosphate during the stationary phase of growth. 1 31 P NMR analysis demonstrated that polyphosphate was accumulated inside the cells during the first 2 or 3 days of growth on minimal media and was subsequently released into the broth. During this study, 31 P NMR analysis revealed the presence of another phosphorus-containing compound (1), resonating at δ -1.07 ppm, which did not correspond to any known material. During growth on minimal medium, a portion of 1 was also found in the culture broth. In this report, we present the molecular structure of 1 using both NMR and MS analysis. Both NMR and biochemical analysis were used to study the cellular distribution of this compound.
Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of c... more Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of considerable biotechnological and therapeutic potential. However, the reducing environment of the cytoplasm inhibits the formation of the intradomain disul®de bonds that are essential for correct folding and functionality of these antibody fragments. Thus, scFvs expressed in the cytoplasm are mostly insoluble and inactive. Here, we describe a general approach for stabilizing scFvs for ef®cient functional expression in the cell cytoplasm in a soluble, active form. The scFvs are expressed as C-terminal fusions with the Escherichia coli maltose-binding protein (MBP). We tested a large panel of scFvs that were derived from hybridomas and from murine and human scFv phage display and expression libraries by comparing their stability and functionality as un-fused versus MBP fused proteins. We found that MBP fused scFvs are expressed at high levels in the cytoplasm of E. coli as soluble and active proteins regardless of the redox state of the bacterial cytoplasm. In contrast, most un-fused scFvs can be produced (to much lower levels) in a functional form only when expressed in trxB À but not in trxB E. coli cells. We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that they perform more ef®ciently in vivo as cytoplasmic intrabodies in E. coli. Thus, MBP seems to function as a molecular chaperone that promotes the solubility and stability of scFvs that are fused to it.
The natural quinone, II-dihydromenaqulnone-9 (M&(11-H)), from Mycobacferium #Mei has been resolve... more The natural quinone, II-dihydromenaqulnone-9 (M&(11-H)), from Mycobacferium #Mei has been resolved into two components by adsorption thin layer chromatography. Chemical analysis of the separated products indicated that they were both 1,4-naphthoquinones of the MKe(II-H) type, differing only in the geometry of the double bond in the ring-terminal isoprene unit. By nuclear magnetic resonance spectroscopy it was possible to show that the major component was all-trans-M&@-H),
About 600 compounds were screened as possible carbon or nitrogen sources for Salmonella typhimuri... more About 600 compounds were screened as possible carbon or nitrogen sources for Salmonella typhimurium LT-2. About 100 utilizable compounds were found.
Transport properties of membrane vesicles isolated from two adenosine triphosphatase-deficient mu... more Transport properties of membrane vesicles isolated from two adenosine triphosphatase-deficient mutants of Escherichia coli, NR70 and DL54, were compared with those of vesicles prepared from the corresponding parental strains. As reported previously (Rosen, 1973; Altendorf et al., 1974), vesicles prepared from these mutants grown under aerobic conditions exhibited defective amino acid transport, and activity was restored after treatment with dicyclohexylcarbodiimide. In sharp contrast, however, vesicles isolated from the same mutants grown anaerobically in the presence of nitrate exhibited completely normal transport activity when assayed under either anaerobic or aerobic conditions. Suppression of the transport defect was not due to the manner by which the vesicles were prepared, and the adenosine triphosphatase deficiency was not ameliorated by anaerobic growth in the presence of nitrite. Finally, the transport activity of vesicles prepared from the mutants grown under aerobic cond...
Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1
Gene, 1989
A putative esterase gene (est) from Acinetobacter calcoaceticus RAG-1 has been cloned into Escher... more A putative esterase gene (est) from Acinetobacter calcoaceticus RAG-1 has been cloned into Escherichia coli. Esterase-positive clones exhibited high levels of esterase activity even in intact cells. In addition, expression of the est gene conferred on E. coli the ability to grow on ...
Sergei Winogradsky, was born in Russia in 1856 and was to become a founder of modern microbiology... more Sergei Winogradsky, was born in Russia in 1856 and was to become a founder of modern microbiology. After his Master's degree work on the nutrition and growth physiology of the yeast Mycoderma vini at the University of St. Petersburg, he joined the laboratory of Anton DeBary in Strassburg. There he carried out his studies on the sulfur-oxidizing bacterium Beggiatoa which resulted in his formulation of the theory of chemolithotrophy. He then joined the Swiss Polytechnic Institute in Zurich where he did his monumental work on bacterial nitrification. He isolated the first pure cultures of the nitrifying bacteria and confirmed that they carried out the separate steps of the conversion of ammonia to nitrite and of nitrite to nitrate. This led directly to the concept of the cycles of sulfur and nitrogen in Nature. He returned to Russia and there was the first to isolate a free-living dinitrogen-fixing bacterium. In the flush of success, he retired from science and spent 15 years on his familial estate in the Ukraine. The Russian revolution forced him to flee Russia. He joined the Pasteur Institute in Paris where he spent his remaining 24 years initiating and developing the field of microbial ecology. He died in 1953.
The est gene encoding an esterase from Acinetobacter lwoffii RAG-1 was cloned into E. coli under ... more The est gene encoding an esterase from Acinetobacter lwoffii RAG-1 was cloned into E. coli under the control of the PL promoter of the phage h. The N-terminal sequence of the first 20 amino acids of the heterolgous expressed esterase corresponded to that obtained from the nucleotide sequence. Antibodies prepared against the over-expressed recombinant esterase in E. coli were used to locate the enzyme primarily in the membrane fractions of A. lwoffiiRAG-1. Comparison with homologous proteins from both eukaryotic and prokaryotic organisms suggest that the RAG-1 esterase exhibits sequence motifs characteristic of both serine proteases and of lipases.
The primary sequence of esterases from Acinetobacter lwoffii RAG‐1 and A. calcoaceticus BD413 wer... more The primary sequence of esterases from Acinetobacter lwoffii RAG‐1 and A. calcoaceticus BD413 were compared with linearized structural sequences of two hundred proteins selected from Brookhaven Protein DataBank using a modified version of the Bowie et al. algorithm [3]. Significant structural homology was found to α/β proteins and specifically to those with the α/β‐hydrolase fold for which the crystal structure was reported. No such homology was detected using common primary sequence alignment programs such as FASTA or BLAST.
Proceedings of the National Academy of Sciences of the United States of America, Jul 1, 1974
A procedure for the purification of Mg2+- Ca2+ adenosinetriphosphatase (EC 3.6.1.3) from E. coli,... more A procedure for the purification of Mg2+- Ca2+ adenosinetriphosphatase (EC 3.6.1.3) from E. coli, yielding relatively large amounts of highly active enzyme, is described. The enzyme consists of four nonidentical subunits. Trypsin treatment of purified enzyme yields a preparation consisting exclusively of the two larger sub- units, which are sufficient for ATPase activity. Purified enzyme is inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3diazole; this inhibition is reversed by dithiothreitol, and the diazole is found preferentially associated with the 13-subunit of the enzyme. Antibody prepared against the trypsin-treated enzyme inhibited various ATP-dependent reactions as well as membrane-bound ATPase itself.
Proceedings of the National Academy of Sciences, 1974
A procedure for the purification of Mg 2+ -Ca 2+ adenosinetriphosphatase (EC 3.6.1.3) from E. col... more A procedure for the purification of Mg 2+ -Ca 2+ adenosinetriphosphatase (EC 3.6.1.3) from E. coli , yielding relatively large amounts of highly active enzyme, is described. The enzyme consists of four nonidentical subunits. Trypsin treatment of purified enzyme yields a preparation consisting exclusively of the two larger subunits, which are sufficient for ATPase activity. Purified enzyme is inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; this inhibition is reversed by dithiothreitol, and the diazole is found preferentially associated with the β-subunit of the enzyme. Antibody prepared against the trypsin-treated enzyme inhibited various ATP-dependent reactions as well as membrane-bound ATPase itself.
Applied and Environmental Microbiology, Mar 1, 1979
The purified extracellular emulsifying factor produced by Arthrobacter RAG-1 (EF-RAG) emulsified ... more The purified extracellular emulsifying factor produced by Arthrobacter RAG-1 (EF-RAG) emulsified light petroleum oil, diesel oil, and a variety of crude oils and gas oils. Although kerosine and gasoline were emulsified poorly by EF-RAG, they were converted into good substrates for emulsification by addition of aromatic compounds, such as 2-methylnaphthalene. Neither aromatic nor aliphatic fractions of crude oil were emulsified by EF-RAG; however, mixtures containing both fractions were emulsified. Pure aliphatic or aromatic hydrocarbons were emulsified poorly by EF-RAG. Binary mixtures containing an aliphatic and an aromatic hydrocarbon, however, were excellent substrates for EF-RAGinduced emulsification. Of a variety of alkylcyclohexane and alkylbenzene derivatives tested, only hexylor heptylbenzene and octylor decylcyclohexane were effectively emulsified by EF-RAG. These data indicate that for EF-RAG to induce emulsification of hydrocarbons in water, the hydrocarbon substrate must contain both aliphatic and cyclic components. With binary mixtures of methylnaphthalene and hexadecane, maximum emulsion was obtained with 25% hexadecane.
Applied and Environmental Microbiology, Jul 1, 1982
Emulsan is an extracellular polymeric bioemulsifier produced by Acinetobacter calcoaceticus RAG-1... more Emulsan is an extracellular polymeric bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. Antibodies prepared against purified emulsan inhibited the activity of the polymer in a standard emulsification test. These antibodies were used to develop a sensitive enzyme-linked immunosorbent assay to monitor changes in cell-free emulsan throughout the growth cycle. This assay was also used to detect emulsan associated with the cell surface and to monitor changes in the distribution of cell-free and cell-associated emulsan throughout the growth cycle. Cells in the early exponential phase exhibited relatively large amounts of cell-associated emulsan which decreased rapidly between the midexponential and early stationary phases. This drop in cell-associated material was accompanied by a rise in the concentration of extracellular polymer. Moreover, in agreement with previous results (C. Rubinovitz, D. L. Gutnick, and E. Rosenberg, manuscript in press), production of cell-free emulsan was enhanced in the presence of chloram- phenicol. The release of this material from the cell surface in the presence of chloramphenicol apparently involved the synthesis of cell-associated cross- reacting material since the relative amount of such cell-bound polymer remained constant during the treatment with the drug.
An esterase activity has been found, both in the cell-free growth medium and on the cell surface ... more An esterase activity has been found, both in the cell-free growth medium and on the cell surface of the hydrocarbon-degrading Acinetobacter calcoaceticus RAG-1. The enzyme catalyzed the hydrolysis of acetyl and other acyl groups from triglycerides and aryl and alkyl esters. Emulsan, the extracellular heteropolysaccharide bioemulsifier produced by strain RAG-1, was also a substrate for the enzyme. Gel filtration showed that the cell-free enzyme was released from the cell surface either emulsan free or associated with the bioemulsifier. The partially purified enzyme was found to interact specifically with the esterified fully active emulsan, but not with the deesterified polymer. A role for esterase in emulsan release from the cell surface was indicated when the enzyme was preferentially depleted from the cell surface under conditions in which emulsan was not released. Such cells lost the capacity to release the biopolymer.
Emulsan is an extracellular polymeric bioemulsifier produced by Acinetobacter calcoaceticus RAG-1... more Emulsan is an extracellular polymeric bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. Antibodies prepared against purified emulsan inhibited the activity of the polymer in a standard emulsification test. These antibodies were used to develop a sensitive enzyme-linked immunosorbent assay to monitor changes in cell-free emulsan throughout the growth cycle. This assay was also used to detect emulsan associated with the cell surface and to monitor changes in the distribution of cell-free and cell-associated emulsan throughout the growth cycle. Cells in the early exponential phase exhibited relatively large amounts of cell-associated emulsan which decreased rapidly between the midexponential and early stationary phases. This drop in cell-associated material was accompanied by a rise in the concentration of extracellular polymer. Moreover, in agreement with previous results (C. Rubinovitz, D. L. Gutnick, and E. Rosenberg, manuscript in press), production of cell-free emulsan ...
Uploads
Papers by David Gutnick