Environmental control of protein export of Bacillus licheniformis NCIB 6346 reveals two types of extracellular proteins

Teichuronic acid was the major anionic polymer of Bacillus licheniformis NCIB 6346 durign phosphate-limited (P-limited) growth in the chemostat. This polymer was also present in significant quantities when B. licheniformis was grown under carbon-limited (C-limited) or magnesium-limited (Mg-limited) conditions where teichoic acid predominated in the cell wall. However, the cell wall composition was not of significance in protein export and the parameters for the excretion process were found to be environmental. In particular, two types of extracellular proteins were identified: the first type of enzyme, penicillinase, was only weakly catabolite repressed; was maximally synthesized and secreted during P-limited growth; was unaffected by growth in high Na⁺ media but its production was inhibited by gramicidin. The second type of enzyme, -amylase, was strongly catabolite repressed and its export was markedly inhibited during P-limited growth or in the presence of Na⁺ or gramicidin. It is noteworthy that the penicillinase carries a glyceride-cysteine modification at its N-terminus whilst the -amylase does not.     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU.     

On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge. Examples of selection-induced mutations have been reported both in bacteria and in yeast. I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed. Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells. Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell. I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.      

A simplified method for the measurement of urinary free cortisol using LC-MS/MS

The measurement of 24 h urinary free cortisol is used in the investigation of patients with symptoms of hypercortisolism. Many different methods have been published for the measurement of cortisol, but most of these methods involve cumbersome pre-extraction of the cortisol prior to analysis. We have developed a method using in-well protein precipitation which serves to clean up the sample without requiring lengthy sample preparation. A Shimadzu SIL-HT autosampler was used to inject 50 microL of extract onto a Phenomemex Gemini C18 guard column attached to a Waters Xbridge C18 column. The eluant was introduced directly into a Waters Quattro Micro tandem mass spectrometer. The method was found to be linear up to 3448 nmol/L with a lower limit of detection of 5.3 nmol/L. Precision and accuracy were acceptable, and no interference was noted from compounds such as prednisolone or fenofibrate. This assay was compared to a previously published method, which uses solid phase extraction prior to LC-MS/MS analysis. We have developed a simplified, robust assay for the quantitation of urinary free cortisol that will increase the throughput of the assay and avoid the use of neurotoxic solvents such as dichloromethane.     

Extended Survival and Persistence of Campylobacter spp. in Water and Aquatic Biofilms and Their Detection by Immunofluorescent-Antibody and -rRNA Staining

In water microcosm experiments, the survival times of Campylobacter isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10°C (202 and 176 h, respectively) than at 22 and 37°C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora, Campylobacter isolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4°C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30°C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4°C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with a Campylobacter-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

Inactivation of Influenza A Virus on Copper versus Stainless Steel Surfaces

Influenza A virus particles (2 × 10⁶) were inoculated onto copper or stainless steel and incubated at 22°C at 50 to 60% relative humidity. Infectivity of survivors was determined by utilizing a defined monolayer with fluorescent microscopy analysis. After incubation for 24 h on stainless steel, 500,000 virus particles were still infectious. After incubation for 6 h on copper, only 500 particles were active.