Gram-negative Bacterium Serratia Marcescens Research Articles

The N Terminus of the HasA Protein and the SecB Chaperone Cooperate in the Efficient Targeting and Secretion of HasA via the ATP-binding Cassette Transporter

Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens. We reconstituted the HasA secretion pathway in Escherichia coli. In E. coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA. The secretion of the isolated C-terminal secretion signal was not SecB-dependent. We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal. Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent. The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein. We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter. Here we show that this abortive interaction between HasA and the E. chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA. N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system. SecB also interacted specifically in vitro with the ABC protein of the Prt system. Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system. We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.

Functional characterization of the HasA(PF) hemophore and its truncated and chimeric variants: determination of a region involved in binding to the hemophore receptor.

Hemophores are secreted by several gram-negative bacteria (Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Yersinia pestis) and form a family of homologous proteins. Unlike the S. marcescens hemophore (HasA(SM)), the P. fluorescens hemophore HasA(PF) has an additional region of 12 residues located immediately upstream from the C-terminal secretion signal. We show that HasA(PF) undergoes a C-terminal cleavage which removes the last 21 residues when secreted from P. fluorescens and that only the processed form is able to deliver heme to the S. marcescens outer membrane hemophore-specific receptor, HasR(SM). Functional analysis of variants including those with an internal deletion of the extra C-terminal domain show that the secretion signal does not inhibit the biological activity, whereas the 12-amino-acid region located upstream does. This extra domain may inhibit the interaction of the hemophore with HasR(SM). To localize the hemophore regions involved in binding to HasR, chimeric HasA(PF)-HasA(SM) proteins were tested for biological activity. We show that residues 153 to 180 of HasA(PF) are necessary for its interaction with the receptor.

NMR studies of the C-terminal secretion signal of the haem-binding protein, HasA.

HasA is a haem-binding protein which is secreted under iron-deficiency conditions by the gram-negative bacterium Serratia marcescens. It is a monomer of 19 kDa (187 residues) able to bind free haem as well as to capture it from haemoglobin. HasA delivers haem to a specific outer-membrane receptor HasR and allows the bacteria to grow in the absence of any other source of iron. It is secreted by a signal peptide-independent pathway which involves a C-terminal secretion signal and an ABC (ATP-binding cassette) transporter. The C-terminal region of the secretion signal containing the essential secretion motif is cleaved during or after the secretion process by proteases secreted by the bacteria. In this work, we study by 1H NMR the conformation of the C-terminal extremity of HasA in the whole protein and that of the isolated secretion signal peptide in a zwitterionic micelle complex that mimicks the membrane environment. We identify a helical region followed by a random-coil C-terminus in the peptide-micelle complex and we show that in both the whole protein and the complex, the last 15 residues containing the motif essential for secretion are highly flexible and unstructured. This flexibility may be a prerequisite to the recognition of HasA by its ABC transporter. We determine the cleavage site of the C-terminal extremity of the protein and analyse the effect of the cleavage on the haem acquisition process.

Purification and characterization of an extracellular heme-binding protein, HasA, involved in heme iron acquisition.

Many bacterial hemoproteins involved in heme acquisition have been isolated recently, comprising outer membrane receptors and extracellular heme-binding protein. The mechanisms by which these proteins extract heme have not been described up to now. One such protein, HasA, which can bind free heme as well as capture it from hemoglobin, is secreted by the Gram-negative bacteria Serratia marcescens under iron deficiency conditions. The fact that HasA does not present sequence similarities with other known hemoproteins suggests that it possesses a new type of heme binding site. This work describes the main physicochemical properties of HasA, essential for understanding its function. HasA is a monomer of 19 kDa that binds one b heme per molecule with high affinity. The electron paramagnetic resonance spectra indicate that the heme iron is in a low-spin ferric state and that the two iron axial ligands are His and His-. The low oxidation-reduction potential value (-550 mV vs standard hydrogen electrode) of the heme bound to HasA suggests that heme could be exposed to the solvent. According to circular dichroism data, the binding of heme does not seem to modify the conformation of HasA.

Bacterial lipases for biotechnological applications

Lipase genes originating from the Gram-negative bacteria Serratia marcescens and Pseudomonas aeruginosa were cloned. S. marcescens lipase was overexpressed in Escherichia coli yielding inclusion bodies which were purified and finally refolded to give enzymatically active lipase. The lipase operon of P. aeruginosa consisting of genes lipA and lipH was cloned behind the T7 φ10 promoter and overexpressed in a lipase-negative P. aeruginosa strain carrying a chromosomal insertion of the gene encoding T7 RNA polymerase. A 3D structural model was built for P. aeruginosa lipase using the coordinates of the Burkholderia cepacia lipase structure which has recently been solved in its open conformation by X-ray crystallography. Both lipases have been purified to homogeneity and were tested for their potential to catalyze biotechnologically important reactions. S. marcescens lipase stereoselectively hydrolyzed racemic isopropylideneglycerol acetate which is a basic building block in a variety of organic synthesis reactions. P. aeruginosa lipase was successfully used for kinetic resolution of chiral alcohols and amines giving enantiomeric excess values of ≥ 95% at reaction rates of 40–50%. Our results demonstrate that both lipases can be produced at levels of 100 mg/l for S. marcescens and 150 mg/l for P. aeruginosa. The recombinant lipase proteins are promising candidates for biotechnological applications.

Simulation of electrostatic and hydrodynamic properties of Serratia endonuclease.

We analyze the electrostatic and hydrodynamic properties of a nuclease from the pathogenic gram-negative bacterium Serratia marcescens using finite-difference Poisson-Boltzmann methods for electrostatic calculations and a bead-model approach for diffusion coefficient calculations. Electrostatic properties are analyzed for the enzyme in monomeric and dimeric forms and also in the context of DNA binding by the nuclease. Our preliminary results show that binding of a double-stranded DNA dodecamer by nuclease causes an overall shift in the charge of the protein by approximately three units of elementary charge per monomer, resulting in a positively charged protein at physiologic pH. In these calculations, the free enzyme was found to have a negative (-1 e) charge per monomer at pH 7. The most dramatic shift in pKa involves His 89 whose pKa increases by three pH units upon DNA binding. This shift leads to a protonated residue at pH 7, in contrast to the unprotonated form in the free enzyme. DNA binding also leads to a decrease in the energetic distances between the most stable protonation states of the enzyme. Dimerization has no significant effect on the electrostatic properties of each of the monomers for both free enzyme and that bound to DNA. Results of hydrodynamic calculations are consistent with the dimeric form of the enzyme in solution. The computed translational diffusion coefficient for the dimer model of the enzyme is in very good agreement with measurements from light scattering experiments. Preliminary electrooptical calculations indicate that the dimer should possess a large dipole moment (approximately 600 Debye units) as well as substantial optical anisotropy (limiting reduced linear electric dichroism of about 0.3). Therefore, this system may serve as a good model for investigation of electric and hydrodynamic properties by relaxation electrooptical experiments.

Crystal Structure of the 50 Kda Metallo Protease from Serratia marcescens

The crystal structure of the 50 Kda metalloprotease from the Gram-negative bacterium Serratia marcescens has been solved and refined to a crystallographic R-factor of 0·192 at 1·80 Å resolution. The structure is very similar to that of alkaline protease from Pseudonomas aeruginosa , in particular the calcium binding "parallel β roll" motif is completely conserved. The N-terminal proteolytic domain shows the typical "metzincin" fold. The active sites of the two enzymes are slightly different, Tyr216 is a Zn ligand in the Serratia metallo protease. The loops 70-77 and 122-132, which encompass the active site cleft, differ due to insertions and deletions so that the Serratia metallo protease seems to have a more open site than the alkaline protease.

Effects of nisin on growth of bacteria attached to meat.

Nisin had an inhibitory effect on gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, and Streptococcus lactis) but did not have an inhibitory effect on gram-negative bacteria (Serratia marcescens, Salmonella typhimurium, and Pseudomonas aeruginosa) attached to meat. Nisin delayed bacterial growth on meats which were artificially inoculated with L. monocytogenes or Staphylococcus aureus for at least 1 day at room temperature. If the incubation temperature was 5 degrees C, growth of L. monocytogenes was delayed for more than 2 weeks, and growth of Staphylococcus aureus did not occur. We also found that the extractable activity of nisin decreased rapidly when the meats were incubated at ambient temperatures and that this decrease was inversely related to the observed inhibitory effect. These findings disclosed that nisin delays the growth of some gram-positive bacteria attached to meat. However, nisin alone may not be sufficient to prevent meat spoilage because of the presence of gram-negative and other nisin-resistant gram-positive bacteria.

Molecular cloning and characterization of a genetic region from Serratia marcescens involved in DNA repair.

We report here the molecular isolation of a DNA fragment which encodes Tag-like activity from the Gram-negative bacterium Serratia marcescens. A recombinant plasmid encoding Tag-like activity was isolated from a S. marcescens plasmid gene library by complementation of an Escherichia coli tag mutant, which is deficient in 3-methyladenine DNA glycosylase I. The clone complements E. coli tag, recA, alkA, but not alkB, mutants for resistance to the DNA-damaging agent methyl methanesulphonate (MMS). The coding region of the Tag activity, initially isolated on a 6.5kb BamHI fragment, was defined to a 1.8kb BglII-SmaI fragment. Labelling of plasmid-encoded proteins using maxicells revealed that the 1.8kb fragment encodes two proteins of molecular weights 42,000 and 16,000. Data presented here suggest that the cloned fragment encodes a DNA repair protein(s) that has similar activity to the 3-methyladenine DNA glycosylase I of E. coli.

Role of the Zinc Atom in the Activity and Conformational Stability of Serratial 56K Protease. A Fluorometric Study1

We found a zinc content of 1 atom per mol in 56K protease (produced by the gram-negative bacterium Serratia marcescens kums 3958) by the neutron activation method. Selective removal of the functional zinc ion from 56K protease with 10 mM tetraethylenepentamine (TEP) in the presence of 10 mM CaCl2 at room temperature at pH 8.0 resulted in complete loss of the enzyme activity without affecting the lambda max of the enzyme, but with a 10% decrease in fluorescence intensity. Ethylenediaminetetraacetic acid (EDTA) at 10 mM gave similar results at pH 5.0, but produced a 30% decrease in fluorescence intensity. Melting profile experiments carried out by monitoring the fluorescence intensity at 333 nm showed a melting temperature (Tm) of about 61.0, 62.5, and 60.0 degrees C at pH 5.0, 7.0, and 8.0, respectively. Tm became much lower upon removal of the zinc ion, falling to 42.5 degrees C with 10 mM TEP or to 47.5 degrees C with 10 mM EDTA in the presence of 10 mM CaCl2. The CD data showed very little change in conformation upon removal of the zinc ion under physiological conditions, but the conformation appears to be readily disrupted to a denatured form by changing either temperature or pH or by exposure to denaturants. These results suggest that a single zinc ion is essential for the enzyme function and contributes to the conformational integrity of the enzyme, but tryptophan residues appear to be not directly related to the enzyme activity.

Effect of Bacterial Endotoxin on Trypanosoma lewisi Infections in Rats

A study was made of the effect of endotoxin on the resistance of rats to Trypanosoma lewisi. The effect on the defense mechanism of the rat was measured by a study of the parasite population during infections. Small serial doses of endotoxin given before the inoculation of trypanosomes increased the resistance of rats, while a single dose given before or simultaneously with the trypanosomes enhanced the infection. The length of time the rats were exposed to serial doses of endotoxin before challenge with trypanosomes appeared to have affected the degree of parasitemia at the peak of the infection. Endotoxin had no effect upon the period of incubation. The time of incubation varied according to the number of trypanosomes inoculated. The larger the inoculum, the shorter the incubation period. There are many reports in the literature indicating that bacterial endotoxin may augment nonspecific defense mechanisms of the host. The chemical and physical nature of endotoxin and methods for its preparation are discussed in several reviews (Bennet and Beeson, 1950; Burrows, 1951; Thomas, 1954, 1958; Halpern, 1959; Rosen, 1961; Whitby et al., 1961). The purpose of this study was to investigate the effect of endotoxin from gram-negative bacteria (Serratia marcescens) on the natural defense mechanisms of the rat against Trypanosoma lewisi. MATERIALS AND METHODS Maintenance of trypanosomes The strain of Trypanosoma lewisi used for this study was obtained from Dr. M. G. Lysenko, University of Wisconsin. Unless otherwise stated, all experiments were performed on adult male Wistar rats weighing between 200 and 300 g. The rats were fed Purina Laboratory Chow pellets and water ad lib. They were housed in standard laboratory animal cages at room temperature. The rats were infected intravenously with trypanosomes from donor rats with 7-day-old infections. Infected blood was obtained from the inferior vena cava of anesthetized donor rats. The red blood cells were removed by centrifugation at 600 rpm for 15 min. The parasites were separated from the plasma, washed with saline, resuspended, Received for publication 2 December 1964. * From a dissertation submitted to the Graduate Faculty of New York University in partial fulfillment of the requirements for the degree of Doctor of Philosophy (1963). t Present address: Department of Microbiology and Parasitology, Universidad Nacional Aut6noma de Mexico, Apartado 20372, Mexico 20, D.F. and counted with a hemocytometer. Dilutions were then made with normal saline until the desired concentrations were obtained for injection into experimental rats. The number of trypanosomes used for individual experiments is given in Table I. During the course of the infection parasite counts were made daily from the day flagellates first appeared in the blood until 3 to 4 days after the peak of infection had been reached. The diluting fluid used for counting the red blood cells and parasites was Hayem's solution (Seiverd, 1952). Blood smears were fixed in absolute methanol for at least 21/ min, air-dried, and stained with Giemsa solution buffered at pH 7.0 (Speirs et al., 1962).

Interaction of an endotoxin with cationic macromolecules.

SUMMARY The interaction of the anionic lipopolysaccharide complex isolated from a Gram-negative bacterium (Serratia marcescens) was studied in aqueous and saline solution with several cationic macromolecules, with the object of selectively inhibiting certain of the biological activities of this polysaccharide endotoxin. Incubation with lysozyme decreased the pyrogenic activity, while the tumour-damaging ability of this polysaccharide remained high. This was in contrast to interaction with RNase and with polymyxin B, in which cases the tumour-damaging activity was decreased while the pyrogenic activity was not affected. Ultracentrifuge experiments indicated that both the tumour-necrotic and especially the fever-producing fractions of this bacterial polysaccharide preparation are of much higher molecular weight (> 10S) than the major component (3.4S), which is either inactive or of low activity. The highest molecular weight fever-producing components appeared to be broken down in an enzymic type of process by the lysozyme.