Amos B. Oppenheim

The Thylakoid FtsH Protease Plays a Role in the Light-Induced Turnover of the Photosystem II D1 Protein

The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate— functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease

Chitin, the second most abundant polysaccharide on earth, is degraded by chitinases and chitobiases. The structure of Serratia marcescens chitobiase has been refined at 1.9 Å resolution. The mature protein is folded into four domains and its active site is situated at the C-terminal end of the central (βα)8-barrel. Based on the structure of the complex with the substrate disaccharide chitobiose, we propose an acid-base reaction mechanism, in which only one protein carboxylate acts as catalytic acid, while the nucleophile is the polar acetamido group of the sugar in a substrate-assisted reaction. The structural data lead to the hypothesis that the reaction proceeds with retention of anomeric configuration. The structure allows us to model the catalytic domain of the homologous hexosaminidases to give a structural rationale to pathogenic mutations that underlie Tay–Sachs and Sandhoff disease.

Differential mRNA stability of the cspA gene in the cold-shock response of Escherichia coli

Exposure of bacterial cells to temperature changes induces the synthesis of a set proteins. We investigated the control of expression of the cspA gene, coding for the major cold‐shock protein of Escherichia coli. This protein was shown to be transiently induced upon shift to low temperature. We demonstrated that the cspA mRNA is extremely unstable at 37°C with a half‐life of approx. 10 s. Upon shift to 15°C cspA mRNA becomes highly stable. This mRNA stability is transient and is lost once the cells are adapted to the low temperature. Transcription fusions of lacZ containing part or most of the cspA gene do not show the rapid degradation at high temperature. Our results suggest that mRNA stability plays a major role in the control of the cspA gene. The expression of cspA is also regulated, to a smaller extent, by the relative increase in transcription after transfer to low temperature. A model by which cspA mRNA is regulated in response to temperature shift is discussed.

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

The bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently to recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a co‐electroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.

Increased Bending Rigidity of Single DNA Molecules by H-NS, a Temperature and Osmolarity Sensor

Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed lambda-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15-20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32 degrees C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.

Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli.

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage λ CII transcriptional activator plays a key role in the lysis‐lysogeny decision by phage λ. Here we demonstrate that the E. coli ATP‐dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat‐shock transcription factor σ32. Proteolysis of σ32 requires, in vivo, the presence of the DnaK‐DnaJ‐GrpE chaperone machine. Neither DnaK‐DnaJ‐GrpE nor GroEL‐GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP‐dependent proteolysis of CII in vitro. The degradation of CII is at least 10‐fold faster than that of σ32. Electron microscopy revealed that purified FtsH forms ring‐shaped structures with a diameter of 6–7 nm.

New components of the chitinolytic system of Trichoderma harzianum

Trichoderma harzianum is a mycoparasite known as a biocontrol agent of several economically important plant pathogenic fungi. Trichoderma spp. utilize chitinolytic enzymes, along with β-1,3-glucanase, to degrade the pathogens cell walls and thus reduce disease level. We employed a set of three fluorescent substrates to identify chitinolytic activities of proteins renatured following their separation by electrophoresis. The chitinolytic system of T. harzianum was found to be more complex than previously reported, consisting of six distinct enzymes, two of which are described here for the first time. The system is composed of two β-1,4 N -acetylglucosaminidases (CHIT102 and CHIT73), and four endochitinases (CHIT52, CHIT42, CHIT33 and CHIT31). The newly described enzymes are CHIT73 and CHIT52. All the chitinolytic enzymes were induced and excreted during growth of Trichoderma on chitin as the sole carbon source. Only CHIT102 was expressed intracellulary, at a low constitutive level, when Trichoderma was grown on glucose. Polyclonal antibodies raised against a purified 41-kDa endochitinase produced by T. harzianum strain P1, reacted only with our CHIT42, suggesting that serologically all the other chitinolytic enzymes are not closely related to the 41-kDa endochitinase. The complexity and diversity of the chitinolytic system of T. harzianum involves the complementary modes of action of six enzymes, all of which are apparently required for maximum efficiency against a broad spectrum of chitin-containing plant pathogenic fungi.

Expression of the genes coding for the Escherichia coli integration host factor are controlled by growth phase, rpoS, ppGpp and by autoregulation

Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth‐phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the rel4 and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth‐phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS.

Compaction of single DNA molecules induced by binding of integration host factor (IHF)

We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force–extension measurements of λ DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA–IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA–IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA–IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.

Mechanisms of biocontrol of soil-borne plant pathogens by Rhizobacteria

Bacterial antagonism, responsible for biological control, may operate by antiobiosis, competition or parasitism. Parasitism relies on lytic enzymes for the degradation of cell walls of pathogenic fungi. Serratia marcescens was found to be an efficient biocontrol agent of Sclerotium rolfsii and Rhizoctonia solani under greenhouse conditions. Populations of 105 or 106 colony forming units g-1 soil were the most effective. Drench and drip application of S. marcescens suspension were more effective in controlling S. rolfsii than spraying, mixing in soil or seed coating. The highest population density of the bacteria in the rhizosphere was found on the proximal portion of the root, decreasing significantly until the tips, where it increased again. The isolated Serratia, found to possess chitinolytic activity, was able to release N-acetyl D-glucosamine from cell walls of S. rolfsii. The gene coding for chitinase was cloned into Escherichia coli and the enzyme was uniquely excreted from the bacterium into its growth medium. When S. rolfsii was sprayed by partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was effective in reducing disease incidence caused by S. rolfsii in beans and R. solani in cotton, under greenhouse conditions. A similar effect was obtained when a viable E. coli cell, containing the plasmid with the chitinase gene (pLCHIA), was applied. It appears that genetic engineering of the lytic enzymes, such as chitinase which play an important role in plant disease control, may improve the efficacy of biocontrol agents.