Characteristics of Escherichia coli grown in bay water as compared with rich medium.

Membrane-filtered bay water can support a certain degree of growth of Escherichia coli organisms isolated from the bay water or from sewage. The effect of the growth medium (bay water versus rich medium) on sensitivities to antimicrobial agents and cell envelope proteins was studied in many of these strains. Bay water-grown cells were less sensitive to bacteriophages and colicins, but were more sensitive to heavy metals and detergents as compared with rich-medium-grown cells. These results indicated that the cell envelope composition of the bay water-grown cells could be modified, resulting in altered susceptibility to various antimicrobial agents. An analysis of cell envelope proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cells from rich-medium-grown cultures contained two or three major outer membrane proteins, whereas in bay water-grown cells, the OmpF protein was greatly reduced.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium.

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Comparison of Vibrio parahaemolyticus grown in estuarine water and rich medium

Cell envelope composition and selected physiological traits of Vibrio parahaemolyticus were studied in regard to the Kanagawa phenomenon and growth conditions. Cell envelopes were prepared from cells cultured in Proteose Peptone-beef extract (Difco Laboratories, Detroit, Mich.) medium or filtered estuarine water. Protein, phospholipid, and lipopolysaccharide contents varied with culture conditions. The phospholipids present in the cell envelopes were identified as phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Phosphatidylethanolamine decreased and phosphatidylglycerol increased in cells grown in estuarine water. Profiles of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated numerous protein species, with four to six predominant proteins ranging from 26,000 to 120,000 in molecular weight. The profile of V. parahaemolyticus cell envelope proteins was unique and might be useful in the identification of the organism. Alkaline phosphatase activity was slightly higher in Kanagawa-negative strains and was higher in cells grown in estuarine water than in cells grown in rich laboratory medium. The DNA levels in estuarine water-grown cells increased, while RNA levels and cell volume decreased. Bacteriophage sensitivity typing demonstrated a close intraspecies relationship. Results indicated that Kanagawa-positive and -negative strains were closely related, but they could be grouped separately and may have undergone starvation-related physiological changes when cultured in estuarine water.     

Growth-medium-dependent repair of DNA single-strand and double-strand breaks in X-irradiated Escherichia coli

The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N. J. Sargentini, W. P. Diver, and K. C. Smith, Radiat. Res. 93, 364-380, 1983). In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair. While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair. This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis. Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process. Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast. Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA. With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.     

Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli.

Supplementation of the growth medium with high concentrations of NaCl, KCl, or sucrose caused a drastic change in the ratio of the two peptidoglycan-associated major outer membrane proteins of Escherichia coli K-12 in that the amounts of proteins b and c present in cell envelope preparations decreased and increased, respectively. Kinetic studies showed that, after the osmolarity of the medium was changed, one protein was hardly incorporated into the membrane, whereas the other was incorporated with an increased rate. After about 1.5 to 2 generations, the cell envelopes obtained the b/c ratio characteristic for the new medium, and both proteins were subsequently incorporated in the cell ensured this new ratio. Once proteins b and c were incorporated in the cell envelope, they were not converted into each other by changes in osmolarity of the growth medium.     

Compromised outer membrane integrity in Vibrio cholerae Type II secretion mutants

The type II secretion (T2S) system of Vibrio cholerae is a multiprotein complex that spans the cell envelope and secretes proteins important for pathogenesis as well as survival in different environments. Here we report that, in addition to the loss of extracellular secretion, removal or inhibition of expression of the T2S genes, epsC-N, results in growth defects and a broad range of alterations in the outer membrane that interfere with its barrier function. Specifically, the sensitivity to membrane-perturbing agents such as bile salts and the antimicrobial peptide polymyxin B is increased, and periplasmic constituents leak out into the culture medium. As a consequence, the σ^E stress response is induced. Furthermore, due to the defects caused by inactivation of the T2S system, the Δeps deletion mutant of V. cholerae strain N16961 is incapable of surviving the passage through the infant mouse gastrointestinal tract. The growth defect and leaky outer membrane phenotypes are suppressed when the culture medium is supplemented with 5% glucose or sucrose, although the eps mutants remain sensitive to membrane-damaging agents. This suggests that the sugars do not restore the integrity of the outer membrane in the eps mutant strains per se but may provide osmoprotective functions.     

Translocases: a bacterial tunnel for drugs and proteins

Unrelated translocases extrude proteins or antimicrobial agents across both membranes of the cell envelope in Gram-negative bacteria. The TolC protein links the translocases to the external environment. The recently determined crystal structure of TolC shows how this universal tunnel operates.     

Production of Cell-Bound Proteinase by Lactobacillus bulgaricus and its Location in the Bacterial Cell

Lactobacillus bulgaricus NCDO 1489 produced a single, cell-bound proteinase during growth on nutrient medium at 45°C. Proteinase activity was optimal at 45–50°C and pH 5.2–5.8. and was inhibited by chelating agents. The enzyme was mainly associated with the cell envelope but could be liberated from cells under conditions favouring autolysis or by treatment of the cells with lysozyme. Its relation to the growth of the organism in milk and possible role in formation of fermented milk products are discussed.     

Cell-envelope changes in mutants ofCitrobacter freundii with altered response to colicin A

Spontaneous colicin A-resistant and -tolerant mutants were isolated fromCitrobacter freundii and classified in five different groups on basis of their sensitivity to bacteriocin S6 produced byKlebsiella pneumoniae. One group of colicin A-resistant mutants was extremely sensitive to ampicillin and desoxycholate; one group of colicin-A-tolerant mutants was extremely sensitive to ampicillin, desoxycholate and EDTA. One of the bacteriocin-S6-insensitive mutants showed filament formation in liquid medium. The cell envelope of one representative strain of each group was isolated and fractionated in a cell-wall- and a cytoplasmic-membrane-enriched fraction. Polyacrylamide-gel electrophoresis of these fractions showed that both fractions differ from those of the wild-type strain in the relative amounts of some proteins. The differences in the cell envelope of all mutants concerned for the greater part the same proteins. Data obtained by phospholipid analysis of the cell envelope of the mutants showed no significant differences.     

Clinically Relevant Growth Conditions Alter Acinetobacter baumannii Antibiotic Susceptibility and Promote Identification of Novel Antibacterial Agents

Biological processes that govern bacterial proliferation and survival in the host-environment(s) are likely to be vastly different from those that are required for viability in nutrient-rich laboratory media. Consequently, growth-based antimicrobial screens performed in conditions modeling aspects of bacterial disease states have the potential to identify new classes of antimicrobials that would be missed by screens performed in conventional laboratory media. Accordingly, we performed screens of the Selleck library of 853 FDA approved drugs for agents that exhibit antimicrobial activity toward the Gram-negative bacterial pathogen Acinetobacter baumannii during growth in human serum, lung surfactant, and/or the organism in the biofilm state and compared those results to that of conventional laboratory medium. Results revealed that a total of 90 compounds representing 73 antibiotics and 17 agents that were developed for alternative therapeutic indications displayed antimicrobial properties toward the test strain in at least one screening condition. Of the active library antibiotics only four agents, rifampin, rifaximin, ciprofloxacin and tetracycline, exhibited antimicrobial activity toward the organism during all screening conditions, whereas the remainder were inactive in ≥ 1 condition; 56 antibiotics were inactive during serum growth, 25 and 38 were inactive toward lung surfactant grown and biofilm-associated cells, respectively, suggesting that subsets of antibiotics may outperform others in differing infection settings. Moreover, 9 antibiotics that are predominantly used for the treatment Gram-positive pathogens and 10 non-antibiotics lacked detectable antimicrobial activity toward A. baumannii grown in conventional medium but were active during ≥ 1 alternative growth condition(s). Such agents may represent promising anti-Acinetobacter agents that would have likely been overlooked by antimicrobial whole cell screening assays performed in traditional laboratory screening media.     

Growth of an ovarian cell line of Galleria mellonella and its response to immune-inducing factors

Antibacterial proteins are produced in the reproductive tracts of some insect species. The advent of a pupal ovarian cell line of the lepidopteran Galleria mellonella offered an opportunity for exploring the use of ovarian tissue culture to induce antimicrobial proteins in lieu of the larvae. The ovarian cell growth rates and cell yields were maximized by adjusting Grace's medium to pH 6.5, adding 15% (v/v) qualified heat-inactivated fetal calf serum, and lowering the sucrose concentration to 9.3 g/L. Five cell forms and biochemical profiles of the collective cell types were analyzed throughout the culture growth cycle. The final modified culture medium did not affect morphogenesis, whereas it increased the culture growth rate by 50% and the final cell yield threefold. The molting and immunoprotein-inducing hormone, 20-hydroxyecdysone, increased culture growth rate and altered the levels of cell types A and D. Neither 20-hydroxyecdysone nor the larval immunizing agents, apolipophorin-III or Bacillus subtilis, in combination or alone, induced antibacterial activity. The bacterium did induce immunity in both larval and adult stages.     

Starvation- and Stationary-phase-induced resistance to the antimicrobial peptide polymyxin B in Salmonella typhimurium is RpoS (sigma(S)) independent and occurs through both phoP-dependent and -independent pathways.

A common stress encountered by Salmonella serovars involves exposure to membrane-permeabilizing antimicrobial peptides and proteins such as defensins, cationic antibacterial proteins, and polymyxins. We wanted to determine if starvation induces cross-resistance to the membrane-permeabilizing antimicrobial peptide polymyxin B (PmB). We report here that starved and stationary-phase (Luria-Bertani [LB] medium) cells exhibited ca. 200- to 1,500-fold-higher (cross-)resistance to a 60-min PmB challenge than log-phase cells. Genetic analysis indicates that this PmB resistance involves both phoP-dependent and -independent pathways. Furthermore, both pathways were sigma(S) independent, indicating that they are different from other known sigma(S) -dependent cross-resistance mechanisms. Additionally, both pathways were important for PmB resistance early during C starvation and for cells in stationary phase in LB medium. However, only the phoP-independent pathway was important for P-starvation-induced PmB resistance and the sustained PmB resistance seen in 24-h-C-starved (and N-starved) or stationary-phase cells in LB medium. The results indicate the presence of an rpoS- and phoP-independent pathway important to starvation- and stationary-phase-induced resistance to membrane-permeabilizing antimicrobial agents.     

Antimicrobial factor from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> inhibits <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>, the causative agent of American foulbrood

Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease.     

Distinctive properties of the hepatitis B virus envelope proteins.

Using recombinant adenoviral vectors, we expressed and characterized the large, middle, and major envelope proteins of hepatitis B virus (HBV). Cells infected with the recombinant adenovirus which contained the large envelope gene (HS1.HP) expressed predominantly large envelope and small but detectable quantities of middle (4%) and major (6%) envelope proteins in the cell lysate. No HBV envelope proteins were detected in the culture medium from HS1.HP-infected cells. Cells infected with recombinant adenovirus which contained the middle envelope gene (HS2.HP) expressed and secreted the middle and major envelope proteins in a molar ratio of 3:1. Cells infected with the recombinant adenovirus which contained the major envelope gene (HS.HP) expressed and secreted major envelope proteins. The HBV envelope proteins secreted by cells infected with either HS2.HP or HS.HP were assembled in 22-nm particles, as shown by velocity sedimentation rate determination, buoyant densities, and electron microscopy. Cells coinfected with a recombinant adenovirus which contained the large envelope gene and with either HS2.HP or HS.HP expressed similar quantities of the large, middle, and major envelope proteins in the cell lysates. Secretion of the major and middle envelope proteins was inhibited more than 95% by the presence of the large envelope proteins. These results suggest that differential biosynthesis, transport, and processing of the envelope proteins occur during HBV infection, allowing efficient assembly and secretion of virions and hepatitis B surface antigen particles.     

Sensitivity of environmental microorganisms to antimicrobial agents.

The sensitivity of different microorganisms, considered as typical representatives of the microflora of soil and water, was established to evaluate the influence of the nonmedical use of antimicrobial agents on bacterial ecology. Only seven strains, six chemoorganotrophs and one chemolithotroph, could be considered as relatively sensitive to the 21 antimicrobial compounds tested. The other 29 microorganisms may be regarded as resistant to most antimicrobial agents. Streptomyces were sensitive to high concentrations of active substances. Broad-spectrum antibiotics showed an effect on environmental bacteria similar to that on human pathogens. Cephalothin stimulated the growth of a Chlorella sp. From these experiments, it appears that spilled antimicrobial agents have little chance of causing an alteration in the microbial ecology.     

A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer

Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.     

Proteomic identification of insulin-like growth factor-binding protein-6 induced by sublethal H2O2 stress from human diploid fibroblasts

Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA3 human skin diploid fibroblasts were exposed to a sublethal dose of H2O2, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen alpha1(VI), collagen alpha2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H2O2-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H2O2-treated cells but not from untreated cells. Increased IGFBP-6 production due to H2O2 treatment was confirmed by RT-PCR and Western blot analyses. While H2O2 induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H2O2-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H2O2 treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, L-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo.     

A novel combination of selective agents for isolation of Leptospira species

A novel combination of antimicrobial agents (sulfamethoxazole, 40 μg/mL; trimethoprim, 20 μg/mL; amphotericin B, 5 μg/mL; fosfomycin, 400 μg/mL; and 5-fluorouracil, 100 μg/mL) was developed for selective isolation of leptospires from contaminated samples. The growth of 16 microorganisms considered as possible contaminants during isolation of Leptospira were inhibited by this antimicrobial cocktail. In contrast, the growth of a smaller inoculum (10(1) cells per mL) of 25 Leptospira strains (representing 18 serovars/serogroups of 5 species) was not suppressed by this antimicrobial combination. This cocktail, after being incorporated into Leptospira growth medium (Korthof's), successfully detected leptospires in environmental soil and water. Based on the results, this selective medium has the potential to meet the existing need for an effective selective medium for the isolation of Leptospira.     

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity in Bacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.     

Regulatory factors that determine growth and phenotype of normal human melanocytes

Normal human melanocytes, unlike malignant melanoma cells, required at least three growth-promoting agents, i.e., phorbol ester for protein kinase C activation and the growth factors basic fibroblast growth factor (bFGF) and insulin, for growth in chemically defined W489 medium. Cell growth was further stimulated by addition of agents that increase intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP) to the medium. Among these agents, the pituitary hormones alpha-melanocyte-stimulating hormone (alpha-MSH) and follicle-stimulating hormone were the most potent, whereas bacterial toxins, including cholera, tetanus, and pertussis toxin and their subunits either were less mitogenic or gave variable results depending on the culture tested. Medium containing phorbol ester PMA, growth factors bFGF and insulin (or insulin-like growth factor-I), and synthetic alpha-MSH supported melanocyte growth for more than 5 months with doubling times between 5 and 8 days. Two copper-binding proteins, ceruloplasmin and tyrosinase, were mitogenic when added to medium and ceruloplasmic induced a long bi- to tripolar-shape of cells. Addition of 1 mM dibutyryl cAMP to the medium led to the formation of dendrites in all cells, with an average of 28 extensions per cell. Although cell growth was inhibited by dibutyryl cAMP, cells were not terminally differentiated and continued to proliferate. Dendritic melanocytes showed a 2.2-fold increase in activity of the tyrosine kinase pp60c-src. The induction of dendritic processes in melanocytes by dibutyryl cAMP or sodium butyrate was reversible and appears to reflect the expression of the mature melanocytic phenotype in situ.