Design and application of rRNA-targeted oligonucleotide probes for the dissimilatory iron- and manganese-reducing bacterium Shewanella putrefaciens.

A 16S rRNA-targeted oligonucleotide probe specific for the iron (Fe3+)- and manganese (Mn4+)-reducing bacterium Shewanella putrefaciens was constructed and tested in both laboratory- and field-based hybridization experiments. The radioactively labeled probe was used to detect S. putrefaciens in field samples collected from the water column and sediments of Oneida Lake in New York and its major southern tributary, Chittenango Creek. S. putrefaciens was quantified by (i) hybridization of the probe to bulk RNA extracted from field samples and normalization of the S. putrefaciens-specific rRNA to total eubacterial rRNA, (ii) a colony-based probe hybridization assay, and (iii) a colony-based biochemical assay which detected the formation of iron sulfide precipitates on triple-sugar iron agar. The results of field applications indicated that the three detection methods were comparable in sensitivity for detecting S. putrefaciens in water column and sediment samples. S. putrefaciens rRNA was detected in the surficial layers of the lake and creek sediments, but the levels of S. putrefaciens rRNA were below the detection limits in the lake and creek water samples. The highest concentrations of S. putrefaciens rRNA, corresponding to approximately 2% of the total eubacterial rRNA, were detected in the surficial sediments of Chittenango Creek and at a midlake site where the Oneida Lake floor is covered by a high concentration of ferromanganese nodules. This finding supports the hypothesis that metal-reducing bacteria such as S. putrefaciens are important components in the overall biogeochemical cycling of iron, manganese and other elements in seasonally anoxic freshwater basins.     

Base composition, size and sequence similarities of genoma deoxyribonucleic acids from clinical isolates of Pseudomonas putrefaciens

The mean base compositions of DNA from 27 strains of Pseudomonas putrefaciens, P. rubescens and P. piscicida ranged from 43-4 to 53-2 mol% GC with genome sizes from 3.04 X 10(9) to 4.23 X 10(9) daltons. On the basis of in vitro DNA-DNA binding, estimated spectrophotometrically from initial renaturation rates, P. putrefaciens strains were heterogenous in the extent to which they shared similar nucleotide sequences, and were divided into four DNA homology groups. The DNA characteristics of strains in these groups correlated with several biochemical characteristics that facilitated identification of clinical isolates of P. putrefaciens. The two species P. putrefaciens and P. rubescens appear to be synonymous and none of the four groups of P. putrefaciens was related in DNA sequences to P. pisicida. Pseudomonas putrefaciens should theretofore be retained as a single species and characteristics for identifying the various groups within the species are listed.     

Do stresses encountered during the smoked salmon process influence the survival of the spoiling bacterium Shewanella putrefaciens?

The influence of different treatments (i.e. cold, NaCl, phenol and anaerobiosis) encountered during the smoked salmon process was studied by analysing the survival capacity of two Shewanella putrefaciens strains (CIP 69.29 and J13.1). Our results indicated that only the salt stress was critical for the survival of S. putrefaciens. Nevertheless, both strains of S. putrefaciens grown at low temperatures developed a cross-protection to a lethal NaCl treatment. To our knowledge, this is the first report showing that growth at low temperatures induces cross-protection towards NaCl challenge. Moreover, we observed a significant sensitization by moderate salt concentration to a phenol treatment. From our combined data, we propose that control of S. putrefaciens proliferation could take place during the smoked salmon process rather than during storage of the final product.     

Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue

The interaction between fish spoilage bacteria, Pseudomonas sp. and Shewanella putrefaciens , was investigated using fish extract and fish tissue as model systems. Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S. putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron. Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S. putrefaciens if the number of Pseudomonas was above 10⁸ cfu ml⁻¹. In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S. putrefaciens. The inhibitory effect was, except for one strain of Pseudomonas , not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp. Finally, siderophore-producing Pseudomonas sp. lowered the maximum cell level of S. putrefaciens 1–2 log units from 10⁹ to 10¹⁰ cfu g⁻¹ when the strains were grown on fish muscle blocks at 0°C but the growth rate of S. putrefaciens was not affected.     

Extracellular Nuclease Activity of Fish Spoilage Bacteria, Fish Pathogens, and Related Species

The production of extracellular deoxyribonuclease and ribonuclease by 23 marine and 3 dairy strains of Pseudomonas putrefaciens, 15 strains of fish-pathogenic fluorescent pseudomonads, 38 strains of fluorescent pseudomonads isolated from haddock, and 34 related organisms was determined by an agar plate method. All strains of P. putrefaciens produced both deoxyribonuclease and ribonuclease. Of the other 87 organisms examined, 26.5% produced ribonuclease and 14.5% produced deoxyribonuclease. All organisms which produced deoxyribonuclease also produced ribonuclease. Deoxyribonuclease production by P. putrefaciens is suggested as a useful criterion of identity for members of this intense fish spoilage species.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

Directing the Biosynthesis of Putrebactin or Desferrioxamine B in Shewanella putrefaciens through the Upstream Inhibition of Ornithine Decarboxylase

To manage iron acquisition in an oxic environment, Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (PB) as its native siderophore. In this work, we have established the siderophore profile of S. putrefaciens in cultures augmented with the native PB precursor putrescine and in putrescine-depleted cultures. Compared to base medium, PB increased by two-fold in cultures of S. putrefaciens with 10?mM NaCl and 20?mM exogenous putrescine. In cultures augmented with 1,4-diaminobutan-2-one (DAB), PB decreased with only 0.02-fold PB detectable at 10?mM DAB. As an ornithine decarboxylase (ODC) inhibitor, DAB depleted levels of endogenous putrescine which attenuated downstream PB assembly. Under putrescine-depleted conditions, S. putrefaciens produced as its replacement siderophore the cadaverine-based desferrioxamine B (DFO-B), as characterised by ESI-MS of the Fe(III) -loaded form (m/z(obs) 614.13; m/z(calc) 614.27). A third siderophore, independent of DAB, was observed in low levels. LC/MS Analysis of the Fe(III) -loaded extract gave m/z(obs) 440.93, which, formulated as a 1?:?1 Fe(III) complex with a macrocyclic dihydroxamic acid, comprising one putrescine- and one cadaverine-based precursor (m/z(calc) 440.14). These results show that the production of native PB or non-native DFO-B by S. putrefaciens can be directed though upstream inhibition of ODC. This approach could be used to increase the molecular diversity of siderophores produced by S. putrefaciens and to map alternative diamine-dependent metabolites.     

Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis.

Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole-cell protein profiling and ribotyping, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently defined species S. alga (U. Simidu et al., Int. J. Syst. Bacteriol, 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can be evaluated only if the organism is correctly identified.     

Changes in cell morphology of Listeria monocytogenes and Shewanella putrefaciens resulting from the action of protamine.

Protamine, which is an antibacterial basic peptide, was shown to alter the cell morphology of Listeria monocytogenes and Shewanella putrefaciens. Atomic force microscopy revealed that protamine smoothed the surface of cells, formed holes in the cell envelope, and caused fusion of S. putrefaciens cells. Immunoelectron microscopy of protamine-treated cells of both L. monocytogenes and S. putrefaciens showed great damage to the cell wall and condensation of the cytoplasm. Respiration of the cells was decreased due to treatment with sublethal concentrations of protamine, probably due to leakage or loss of cell envelope potential. It was concluded that protamine disrupted the outer surface structure and condensed the cytoplasm of sensitive cells and, in sublethal concentrations, altered membrane structures, thereby eliminating respiration.     

Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.     

Spoilage of vacuum-packaged dark, firm, dry meat at chill temperatures.

The flora of vacuum-packaged dark, firm, dry meat included thred organisms not usually found on vacuum-packaged meat, Yersinia enterocolitica, Enterobacter liquefaciens, and Alteromonas putrefaciens. Y. enterocolitica did not affect the meat quality. Production of spoilage odors by E. liquefaciens could be prevented by addition of glucose or citrate to the meat. Greening of meat could be prevented by addition of glucose or citrate to the meat. Greening of meat by A. putrefaciens was not prevented by addition of glucose, as the organism degraded cysteine with the release of H2S even when glucose was present. To prevent greening, growth of A. putrefaciens must be inhibited by reducing the meat pH to less than 6.0.     

Spoilage of vacuum-packaged dark, firm, dry meat at chill temperatures.

The flora of vacuum-packaged dark, firm, dry meat included thred organisms not usually found on vacuum-packaged meat, Yersinia enterocolitica, Enterobacter liquefaciens, and Alteromonas putrefaciens. Y. enterocolitica did not affect the meat quality. Production of spoilage odors by E. liquefaciens could be prevented by addition of glucose or citrate to the meat. Greening of meat could be prevented by addition of glucose or citrate to the meat. Greening of meat by A. putrefaciens was not prevented by addition of glucose, as the organism degraded cysteine with the release of H2S even when glucose was present. To prevent greening, growth of A. putrefaciens must be inhibited by reducing the meat pH to less than 6.0.     

Characterisation of a tetrasaccharide released on mild acid hydrolysis of LPS from two rough strains of Shewanella species representing different DNA homology groups

A reducing tetrasaccharide of the following structure was released by mild acid hydrolysis of R-type LPS from Shewanella putrefaciens strains NCIMB 10472 and 10473. The same tetrasaccharide containing acetal-linked open-chain GalNAc is present in the core region of LPS from S. oneidensis strain MR-1 and may be characteristic of genomic groups II and III of S. putrefaciens and related strains. (1S)-d-GalaNAc-(1-->4,6)-alpha-d-Galp-(1-->6)-alpha-d-Galp-(1-->3)-d-Gal.     

一种新的异育银鲫病原———腐败希瓦氏菌

【目的】江苏盐城一家养殖场的异育银鲫暴发疾病,通过对病原进行研究,旨在为该病的防治提供理论依据和参考。【方法】从病鱼体表病灶和内脏中分离出优势菌株,经人工感染试验证实为病原菌。采用传统的形态、生理生化表型鉴定与16S rDNA序列分析相结合的方法确定菌株的分类地位。运用K-B琼脂法对病原菌株进行药物敏感性测定。【结果】综合菌株形态、生理生化表型以及16S rDNA序列分析的结果,确定该分离株为腐败希瓦氏菌(Shewanella putrefaciens)。回接感染试验证实腐败希瓦氏菌即是导致此次异育银鲫发病死亡的致病原,其半数致死量(LD50)为2.1×103cfu/g。该株腐败希瓦氏菌对吡哌酸、萘啶酸、氟哌酸、氟啶酸、氟苯尼考、利福平、美满霉素、氟罗沙星、恩诺沙星、复达欣、菌必治、先锋Ⅳ、罗红霉素和左氟沙星等抗生素敏感。【结论】首次报道了异育银鲫一种新的病原,说明腐败希瓦氏菌作为一种潜在的新病原也可能会对异育银鲫的养殖造成威胁。     

The effect of potassium sorbate on the structural integrity of Alteromonas putrefaciens

Treatment of Alteromonas putrefaciens with potassium sorbate at pH 7.0 resulted in increased hydrophobicity of the cell wall and lysis on exposure to lysozyme. These effects could be overcome to some extent by the addition of magnesium ions to the incubation medium. Transmission and scanning electron microscopy provided evidence of outer membrane damage in sorbate-treated cells. Dissociated sorbate ion was shown to have an inhibitory effect on A. putrefaciens .     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens.

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.     

Extracellular Enzymic Activity of Poultry Spoilage Bacteria

The extracellular enzymic activity has been studied of 224 strains of bacteria isolated mainly at 1° from spoiling chickens and turkeys and from poultry processing plants. The isolates comprised 44 strains of pigmented Pseudomonas , 57 strains of nonpigmented Pseudomonas , 29 strains of Ps. putrefaciens , 50 strains of oxidase positive Acinetobacter and 44 strains of oxidase negative Acinetobacter. None of the strains showed any significant activity against dextrin, starch, glycogen, inulin, dextran, xylan or pectin. Proteolytic activity was found mainly amongst 2 groups of pigmented pseudo-monads, and Ps. putrefaciens. Nuclease activity was found particularly amongst strains of Ps. putrefaciens and the oxidase negative Acinetobacter strains isolated from spoiling poultry. Almost all of the strains showed lipolytic activity when tested with tributyrin and a proportion of strains could also attack chicken fat. This latter property was particularly evident amongst the nonpigmented Pseudomonas strains.     

A preliminary study of the use of Colilert for water quality monitoring

Substrate specificities of proteases produced by two putrefactive marine bacteria, Shewanella putrefaciens and Alteromonas haloplanktis , were surveyed by using peptidyl-7-amino-4-methylcoumarin (MCA-substrates). Shewanella putrefaciens produced trypsin-like enzyme(s) showing broad spectrum specificity and chymotrypsin-like enzyme specifically hydrolysing Glt-Gly-Gly-Phe-MCA. Alteromonas haloplanktis produced high activity of ammopeptidase and trypsin-like enzyme(s) preferring Z-Phe-Arg-MCA, Bz-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA. The two organisms would be able to utilize different proteins for their growth.