Accumulation of Poly (beta-Hydroxybutyrate) by Halobacteria

Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas fragi, Escherichia coli, and Salmonella "anatum" were challenged with butylated hydroxyanisole (BHA). Susceptibility was measured as the concentration of BHA required to cause a 90% reduction in bacterial survivors. Staphylococcus aureus LP and P. fragi were two of the most resistant species examined; C. perfringens and P. fluorescens were the most susceptible. Gram stain reaction was found not to be a strict indicator of bacterial susceptibility to BHA. There was no obvious relationship between individual fatty acids and susceptibility. The ratio of saturated to unsaturated fatty acids in the total lipid fraction of only the gram-positive species was related to susceptibility. The ratios of saturated to unsaturated fatty acids of other fractions were not related to susceptibility.     

Volatile compounds produced by meat pseudomonads and relate reference strains during growth on beef stored in air at chill temperatures

Volatile compounds produced by 31 strains of pseudomonads and by reference strains of Pseudomonas fragi and Ps. fluorescens biotype 1 during growth on beef stored at 6 degrees C in air were analysed by gas chromatography-mass spectrometry of headspace gases. Compounds of major sensory significance were ethyl and methyl esters of C2-C8 fatty acids and sulphur-containing compounds which included methane- and isopropanethiols and their related sulphides and thioesters but not hydrogen sulphide. Ester production was mainly associated with growth of some, but not all, Ps. fragi and related meat strains but sulphur-containing compounds were produced by all but a single meat strain. A minority of other meat strains produced greater amounts of methyl ketones, secondary alcohols and unsaturated hydrocarbons believed to be of lipid origin.     

Volatile compounds produced by meat pseudomonads and related reference strains during growth on beef stored in air at chill temperatures

Volatile compounds produced by 31 strains of pseudomonads and by reference strains of Pseudomonas fragi and Ps. fluorescens biotype 1 during growth on beef stored at 6°C in air were analysed by gas chromatography-mass spectrometry of headspace gases. Compounds of major sensory significance were ethyl and methyl esters of C₂–C₈ fatty acids and sulphur-containing compounds which included methane- and isopropanethiols and their related sulphides and thioesters but not hydrogen sulphide. Ester production was mainly associated with growth of some, but not all, Ps. fragi and related meat strains but sulphur-containing compounds were produced by all but a single meat strain. A minority of other meat strains produced greater amounts of methyl ketones, secondary alcohols and unsaturated hydrocarbons believed to be of lipid origin.     

Survival and Phospholipid Fatty Acid Profiles of Surface and Subsurface Bacteria in Natural Sediment Microcosms

Although starvation survival has been characterized for many bacteria, few subsurface bacteria have been tested, and few if any have been tested in natural subsurface porous media. We hypothesized that subsurface bacteria may be uniquely adapted for long-term survival in situ. We further hypothesized that subsurface conditions (sediment type and moisture content) would influence microbial survival. We compared starvation survival capabilities of surface and subsurface strains of Pseudomonas fluorescens and a novel Arthrobacter sp. in microcosms composed of natural sediments. Bacteria were incubated for up to 64 weeks under saturated and unsaturated conditions in sterilized microcosms containing either a silty sand paleosol (buried soil) or a sandy silt nonpaleosol sediment. Direct counts, plate counts, and cell sizes were measured. Membrane phospholipid fatty acid (PLFA) profiles were quantified to determine temporal patterns of PLFA stress signatures and differences in PLFAs among strains and treatments. The Arthrobacter strains survived better than the P. fluorescens strains; however, differences in survival between surface and subsurface strains of each genus were not significant. Bacteria survived better in the paleosol than in the nonpaleosol and survived better under saturated conditions than under unsaturated conditions. Cell volumes of all strains decreased; however, sediment type and moisture did not influence rates of miniaturization. Both P. fluorescens strains showed PLFA stress signatures typical for gram-negative bacteria: increased ratios of saturated to unsaturated fatty acids, increased ratios of trans- to cis-monoenoic fatty acids, and increased ratios of cyclopropyl to monoenoic precursor fatty acids. The Arthrobacter strains showed few changes in PLFAs. Environmental conditions strongly influenced PLFA profiles.     

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

Selection of pH buffers for use in conductimetric microbiological assays

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5° to 30°C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

A Simple Procedure for Detecting the Presence of Cyclopropane Fatty Acids in Bacterial Lipids

Four gram-negative bacterial species, including Escherichia coli strain B, Serratia marcescens, Pseudomonas fluorescens, and Vibrio cholerae (comma) strain NIH 41, were investigated for fatty acid content by gas-liquid chromatography involving a preparatory technique which facilitated detection of cyclopropane fatty acids. Methyl esters of fatty acids were subjected to mild catalytic hydrogenation to eliminate unsaturates. Hydrogenation was followed by bromination which removed cyclopropane acids from chromatographic profile patterns. Lactobacillic acid (cis-11,12-methyleneoctanoate) and cis-9,10-methylenehexadecanoate, previously reported in lipids of E. coli and S. marcescens, were found in small amounts in P. fluorescens but were not detected in V. cholerae.     

The effects of temperature and growth rate on the proportion of unsaturated fatty acids in bacterial lipids

The effects of temperature and growth rate on the fatty acid composition of the extractable lipids of four mesophilic and three psychotrophic bacteria were examined. Two of the mesophiles (Escherichia coli and Pseudomonas aeruginosa) increased the proportion of unsaturated fatty acids in their lipids with decreasing temperature over their whole growth temperature range. The other mesophiles (Enterobacter aerogenes and Lactobacillus casei) increased the proportion of unsaturated fatty acids with decreasing temperature only over the lower half of their growth temperature ranges. The psychrotrophs Pseudomonas fluorescens and Enterobacter sp. had a constant proportion of unsaturated acids over the lower half of their growth temperature range, while the psychotrophic Lactobacillus sp. showed no consistent change in its unsaturated fatty acid composition with temperature. All species showed some variation of unsaturated fatty acid composition with growth rate at the highest and lowest growth temperatures, although such variations were small in some species (Ent. aerogenes and Lactobacillus sp.).     

Antimicrobial activity of shredded carrot extracts on food-borne bacteria and yeast

Purified ethanolic extracts of peeled and shredded carrots showed an antimicrobial effect against a range of food-borne micro-organisms. The minimum inhibitory concentration, expressed as mg ml^-1 dried carrot material used for the extraction were: Leuconostoc mesenteroides , 27; Listeria monocytogenes , >27 <55; Staphylococcus aureus , >27 <55; Pseudomonas fluorescens , >55 <110; Candida lambica , >55 < 110; Escherichia coli , > 110 <220. The antimicrobial activity was not linked to phenolic compounds but was presumably due to apolar components. Free saturated fatty acid (dodecanoic acid) and methyl esters of saturated fatty acids (of dodecanoic and pentadecanoic acids) were identified in purified active extracts of carrots by gas chromatography coupled to mass spectrometry and could be responsible for the antimicrobial activity. This effect did not seem to play a role in the resistance of shredded carrots to microbial spoilage, although the antimicrobial activity was present in fresh carrots at concentrations sufficient to inhibit spoilage bacteria.     

Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage

A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.     

RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly

The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonasfragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method, respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonasfragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.     

Metabolic activities of pseudomonads in batch cultures in extract of minced lamb

E.H. DROSINOS AND R.G. BOARD. 1994. Pseudomonas fragi, Ps. lundensis and Ps. fluorescens were studied in axenic batch cultures growing in a lamb juice (pH 6.0) aerobically or in an atmosphere ( Ps. fragi only) enriched with carbon dioxide at 4C. With all but a glucose dehydrogenase-deficient strain of Ps. fluorescens there was a sequential catabolism of glucose and lactate. Diauxic growth was observed in a nutrient-deficient meat juice supplemented with glucose and lactate. A transient peak in the concentration of gluconate and pyruvate was associated with the catabolism of glucose and lactate respectively. With Ps. fluorescens deficient in glucose dehydrogenase there was simultaneous catabolism of glucose and lactate. The stereoisomers of lactate were catabolized simultaneously in a laboratory medium. Glucose-6-phosphate was oxidized to 6-phosphogluconate by Ps. fragi and Ps. lundensis under aerobic conditions only. Creatine and creatinine were catabolized by Ps. fragi under aerobic conditions only. There was a slight decrease in the concentration of total amino acids (ninhydrin-reactive material) during the exponential phase of growth. The results suggest that the dominance of Ps. fragi in the climax populations in meat is due to catabolism of amino acid related substrates, creatine and creatinine.     

Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.     

Weak antimicrobial effect of carob (Ceratonia siliqua) extract against food-related bacteria in culture media and model food systems

A weak antibiotic effect of carob (Ceratonia siliqua) extract against Staphylococcus aureus S-6, St. aureus 722, and Pseudomonas fragi, was evident when the strains were screened with the paper disk assay. An intermediate inhibitory action of carob on Listeria monocytogenes, Salmonella enteritidis, and Brochothrix thermosphacta was found with the same technique while Shewanella putrefaciens was not inhibited. The inhibitory action of extract against St. aureus and P. fragi was also confirmed in broth culture while little effect was noted with S. enteritidis. The addition of extract to tuna and potato salad stored under aerobic and modified atmosphere packaging conditions at 4 °C, affected the survival of St. aureus but not that of S. enteritidis.     

Induction of enzymes involved in fatty acid beta-oxidation in Pseudomonas fragi B-0771 cells grown in media supplemented with fatty acid.

Induction of the enzymes involved in fatty acid beta-oxidation in Pseudomonas fragi B-0771 cells grown in a medium containing straight chain saturated fatty acids was studied. The acyl-CoA dehydrogenase (ACDH) activity was induced during the exponential phase in cells grown in palmitic acid-supplemented medium, reached a maximum at the early stationary phase, and then gradually decreased thereafter. Changes in the overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, both existing on the multienzyme complex (HDT) involved in fatty acid beta-oxidation, were similar to that in ACDH activity. Straight chain saturated fatty acids having more than 6 carbon atoms could induce both the ACDH and HDT activities, and C13-C15 fatty acids caused the greatest induction of both activities. Changes in the overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase correlated with that in the amount of the alpha-subunit of HDT during the entire culture period in the medium containing palmitic acid. Surprisingly, the stoichiometry of the alpha- and beta-subunit proteins of HDT was not maintained into the stationary phase culture, though the genes encoding the alpha- and beta-subunits are tandemly coded in bacterial genomic DNA.     

Chemical constituents of some Tricholomataceae

Sterols, fatty acids and free amino acids of four species of Tricholomataceae were investigated. They contained ergosterol in high amount and the related sterols, fucosterol, sitosterol, cholesterol and brassicasterol. Linoleic and palmitic are the main unsaturated and saturated fatty acids respectively. The most abundant free amino acids were alanine, valine and proline.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 micrograms/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na+, K+, Mg2+ or Ca2+ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

Homologous expression of the lipase and ABC transporter gene cluster, tliDEFA, enhances lipase secretion in Pseudomonas spp.

The ABC transporter TliDEF was found to be an efficient secretory apparatus for extracellular lipase TliA in Pseudomonas fluorescens. For the enhanced secretion of the lipase, we tried to coexpress tliA and tliDEF in various Pseudomonas species. Whereas the coexpression of tliA and tliDEF was required for the lipase secretion in P. fragi, the expression of tliA was sufficient for the lipase secretion in P. fluorescens, P. syringae, and P. putida, indicating the existence of compatible ABC transporter in these species. However, P. fluorescens harboring tliDEFA secreted much more lipase than P. fluorescens harboring only tliA, but the tliDEF was functional only at temperatures below 30 degrees C. The recombinant P. fluorescens overexpressing tliDEFA showed the highest secretion level, 217 U/ml. OD (optical density) (28 microg/ml. OD) of lipase in Luria-Bertani medium under microaerated conditions. With the increase of aeration, the lipase production was decreased and the lipase seemed to be degraded as the cells entered the cell death phase. These results demonstrate that P. fluorescens can be used as a host system for the secretory production of the lipase using the ABC transporter, thus producing lipase in over 14% of the total protein.     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.