Characterization of Porins Isolated from the Outer Membrane of Serratia liquefaciens

A major outer membrane protein with an apparent molecular weight of 42 kDa was purified from Serratia liquefaciens grown on Brain Heart Infusion medium. The same protein was obtained when the cells were grown on a synthetic medium supplemented with 2% glucose. The amino acid composition of this protein revealed it to be hydrophilic. The pore-forming ability of the 42-kDa protein was determined by the liposome swelling assay. This assay demonstrated that the protein forms nonspecific channels with a diameter between 1.16 and 1.6 nm. An additional protein with a molecular weight of 47 kDa was obtained on synthetic medium supplemented with maltose. This protein exhibited specific pore-forming ability to maltose and maltodextrins, but was also permeable to other compounds, according to their size. When bacteria were grown on Nutrient Broth medium, two outer membrane proteins with molecular weights of 41 kDa and 42 kDa were produced by the bacteria. All three types of proteins represent monomers of respective oligomers. The monomers did not exhibit pore-forming ability when incorporated into liposomes. We, therefore, propose that the oligomer is the functional unit of a porin capable of forming permeability channels in the outer membrane of Serratia liquefaciens. These results indicate that S. liquefaciens contains several porins exhibiting specific osmoregulation or that are induced by a specific nutrient, where the 42-kDa outer membrane protein of this bacterium is certainly a major porin. Received: 6 July 1998 / Accepted: 19 August 1998     

Characterization of Six Bacteriophages of Serratia liquefaciens CP6 Isolated from the Sugar Beet Phytosphere

Six phages (ΦCP6-1 to ΦCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (ΦCP6-1 and ΦCP6-4). Like ΦCP6-2 and ΦCP6-5, ΦCP6-1 belonged to the family Siphoviridae, while ΦCP6-4 exhibited the morphology of the family Podoviridae. The other phages were members of the family Myoviridae. DNA-DNA cross-hybridization revealed that ΦCP6-1 and ΦCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like ΦCP6-2, ΦCP6-3, and ΦCP6-5, ΦCP6-1 was capable of forming a lysogenic association with its host, while ΦCP6-4 and ΦCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that ΦCP6-4 had a much shorter latent period and a smaller burst size than ΦCP6-1. Also, ΦCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 × 10⁻⁹ to 3.9 × 10⁻⁷, whereas ΦCP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.     

Secretion of Serratia liquefaciens phospholipase from Escherichia coli

The Serratia liquefaciens phospholipase (PhIA) is secreted to the medium from its natural host. Here we present results which indicate that, when cloned and expressed in Escherichia coli, secretion can be mediated by a putative host-encoded pathway, expression of which is controlled by FlhD (formerly FlbB), the master regulator of the flagellar/ chemotaxis regulon. In the absence of this secretion pathway, the synthesized phospholipase accumulates inside the host cell where it forms a complex with the PhlB protein. PhlB, which is encoded from the promoter distal gene of the phospholipase operon, inhibits the phospholipase activity of PhlA. Formation of this enzymatically inactive PhlA/PhlB complex is required for maintenance of cell viability.     

Kinetic mechanism and location of rate-determining steps for aspartase from Hafnia alvei

Coupled spectrophotometric assays that monitor the formation of fumarate and ammonia in the direction of aspartate deamination and aspartate in the direction of fumarate amination were used to collect initial velocity data for the aspartase reaction. Data are consistent with rapid equilibrium ordered addition of Mg2+ prior to aspartate but completely random release of Mg2+, NH4+, or fumarate. In addition to Mg2+, Mn2+ can also be used as a divalent metal with Vmax 80% and a Kaspartate 3.5-fold lower than when Mg2+ is used. Monovalent cations such as Li+, K+, Cs+, and Rb+ are competitive vs. either aspartate or NH4+ but noncompetitive vs. fumarate. A primary deuterium isotope effect of about 1 on both V and V/Kaspartate is obtained with (3R)-L-aspartate-3-d, while a primary 15N isotope effect on V/Kaspartate of 1.0239 +/- 0.0014 is obtained in the direction of aspartate deamination. A secondary isotope effect on V of 1.13 +/- 0.04 is obtained with L-aspartate-2-d. In addition, a secondary isotope effect of 0.81 +/- 0.05 on V is obtained with fumarate-d2, while a value of 1.18 +/- 0.05 on V is obtained by using (2S,3S)-L-aspartate-2,3-d2. These data are interpreted in terms of a two-step mechanism with an intermediate carbanion in which C-N bond cleavage limits the overall rate and the rate-limiting transition state is intermediate between the carbanion and fumarate.     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1529

The following structure of the pentasaccharide repeating unit of an acidic O-polysaccharide of Hafnia alvei PCM 1529 was established by sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy: [Carbohydrate structure: see text].     

Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1546

An acidic O-polysaccharide isolated by mild acid hydrolysis from the lipopolysaccharide of Hafnia alvei PCM 1546 is composed of D-Gal, D-Glc, D-GlcA, D-GalNAc and O-acetyl groups in the ratios 1:1:1:2:1.6. On the basis of sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the pentasaccharide repeating unit of the polysaccharide was established: [see equation in text].     

Pulsed-field gel electrophoresis typing of Hafnia alvei isolated from chub-packed and retail ground beef

Thirty-eight isolates of Hafnia alvei were characterized by biochemical profiles, ribotyping and pulsed-field gel electrophoresis (PFGE) patterns. The isolates were recovered from chub-packed (19 isolates) or retail (nine isolates) ground beef, or were obtained from culture repositories (10 isolates). Biochemical profiling differentiated the 38 isolates into five groups and a commercial ribotyping method recognized 11 groups, whereas PFGE differentiated the same 38 isolates into 19 groups. These data substantiate that PFGE is a highly discriminatory tool for establishing the relatedness among Hafnia alvei strains.     

The Yersinia HPI is present in Serratia liquefaciens isolated from meat

AIMS: The aim of the study was to screen the Enterobacteriaceae flora of meat for the presence of bacteria harbouring the Yersinia high-pathogenicity island (HPI). METHODS AND RESULTS: Bacteria from 29 meat and 29 liver samples were isolated on violet-red bile glucose agar. A total of 197 isolates were screened for the presence of the irp2 gene, encoded within the HPI, by PCR. One isolate that was positive for irp2 gene was also positive for the fyuA, irp1, ybtP/ybtQ, ybtX/ybtS and int/asn tRNA genes by PCR. The presence of fyuA, irp1 and irp2 genes was confirmed by Southern hybridization. CONCLUSIONS: The isolate was identified as Serratia liquefaciens by sequencing of the 16S rRNA gene and by ribotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a Serratia harbouring the Yersinia HPI. Serratia is a frequently occurring Enterobacteriaceae genus in chill-stored meat.     

Antibiotic Susceptibilities of Serratia marcescens and Enterobacter liquefaciens

Production of 5'-nucleotides by Serratia marcescens and Enterobacter liquefaciens correlates with deoxyribonuclease production, indicating the close relationship between these two organisms. To determine further relationships, susceptibilities of 279 strains of the tribe Klebsielleae were determined by the high-potency disc method, agar-dilution method, or both, by using 14 antibiotics. Ninety-seven per cent of S. marcescens (201 of 207 strains) and 100% of E. liquefaciens (17 strains) had minimum inhibitory concentration (MIC) of 100 mug/ml or greater with colistin and polymyxin B. With these two antibiotics, 93% of other Enterobacter species (28 strains) had MIC values of less than 1.6 mug/ml, and 100% of Klebsiella (27 strains) had MIC values less than 1.6 mug/ml. Consistent patterns were not noted with the other antibiotics tested, but the results with colistin and polymyxin B provide additional evidence of the close relationship of S. marcescens and E. liquefaciens.     

Invasion and intracellular survival of Hafnia alvei strains in human epithelial cells

Aims: The aim of this study was to investigate the invasion and intracellular survival of different Hafnia alvei strains in HeLa cells. Methods and Results: We performed different experiments on the bacterial invasion of different strains of H. alvei into the HeLa cell line using gentamicin protection assays and immunofluorescence. We also report the time course of cell internalization and the effects of inhibitors on the invasion of H. alvei. Levels of invasion varied depending on the conditions (strain, time and inoculum size) used. Conclusions: This study revealed that H. alvei strains were able to enter and persist in a human epithelial cell line. Significance and Impact of the Study: Our in vitro findings highlight the possibility that some H. alvei strains may exploit nonprofessional phagocytes or nonphagocytic cells to spread in vivo, which may be important for the persistence and establishment of an asymptomatic carrier state.     

Structure of the lipopolysaccharide core region of Hafnia alvei strains 1185 and 1204

Sugar and methylation analyses using gas chromatography/mass spectrometry and NMR spectroscopy proved that the core oligosaccharides of Hafnia alvei strains 1185 and 1204 have the following formula: carbohydrate sequence [see text] where Kdo = 3-deoxy-oct-2-ulosonic acid and P-PEtN = diphosphorylethanolamine. The structure shown above is a slight modification of the typical core region of H. alvei lipopolysaccharides. The difference refers to one sugar only: terminal galactose is present in the core of strains of 1185 and 1204, while terminal glucose in the typical core.     

Two Kdo-Heptose Regions Identified in Hafnia alvei 32 Lipopolysaccharide: the Complete Core Structure and Serological Screening of Different Hafnia O Serotypes

Hafnia alvei, a gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. Various 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-containing fragments different from known structures of core oligosaccharides were previously found among fractions obtained by mild acid hydrolysis of some H. alvei lipopolysaccharides (LPSs). However, the positions of these segments in the LPS structure were not known. Analysis of de-N,O-acylated LPS by nuclear magnetic resonance spectroscopy and mass spectrometry allowed the determination of the location of a Kdo-containing trisaccharide in the structure of H. alvei PCM 32 LPS. It was established that the trisaccharide {l-α-d-Hepp-(1→4)-[α-d-Galp6OAc-(1→7)]-α-Kdop-(2→} is an integral part of the outer-core oligosaccharide of H. alvei 32 LPS. The very labile ketosidic linkage between →4,7)-α-Kdop and →2)-Glcp in the core oligosaccharide was identified. Screening for this Kdo-containing trisaccharide was performed on the group of 37 O serotypes of H. alvei LPSs using monospecific antibodies recognizing the structure. It was established that this trisaccharide is a characteristic component of the outer-core oligosaccharides of H. alvei 2, 32, 600, 1192, 1206, and 1211 LPSs. The weaker cross-reactions with LPSs of strains 974, 1188, 1198, 1204, and 1214 suggest the presence of similar structures in these LPSs, as well. Thus, we have identified new examples of endotoxins among those elucidated so far. This type of core oligosaccharide deviates from the classical scheme by the presence of the structural Kdo-containing motif in the outer-core region.Lipopolysaccharide (LPS) (endotoxin) is the main surface antigen and an important virulence factor of most of the gram-negative bacteria that are pathogenic for humans and animals (46). LPS contributes greatly to the structural integrity of bacteria and constitutes a “pathogen-associated molecular pattern” for host infection (46). As one of the most potent natural activators of the innate immune system, LPS is recognized by different classes of receptors present on macrophages, monocytes, B and T cells, neutrophils, endothelial cells, and epithelial cells (46). Endotoxins stimulate these cells to produce multiple inflammatory mediators responsible for immunotoxicity (e.g., tumor necrosis factor alpha, interleukin 1 [IL-1], IL-6, IL-8, gamma/alpha interferon, NO, platelet-activating factor, and endorphins). Interaction of LPS with the CD14/Toll-like receptor 4/MD-2 receptor complex constitutes a major mechanism responsible for the innate immune response to infection by gram-negative bacteria (1, 46). A large amount of LPS released into the bloodstream triggers the excessive inflammatory response of the innate immune system, leading to sepsis and septic shock (6). High levels of inflammatory mediators have profound effects on the cardiovascular system, kidneys, lungs, liver, central nervous system, and coagulation system. Following their action, renal failure, myocardial dysfunction, acute respiratory distress syndrome, hepatic failure, and disseminated intravascular coagulation can occur, which may result in death (6). Despite intense research on the etiology and treatment of sepsis, its severe form still carries a high mortality rate (6, 46).Hafnia alvei has been reported to be an opportunistic human pathogen. This gram-negative bacterium and its LPS are among the identified causative agents of bacteremia and septicemia in humans and animals (19). For the years 2001 to 2003, up to 42 cases of H. alvei bacteremia were reported annually in the United Kingdom. Most of them were monomicrobic infections, and in ∼33% of the cases, H. alvei was isolated, not only from the blood, but also from hepatic abscesses, pancreatic pseudocyst fluid, sputum, feces, and central venous catheters (19). Besides bacteremia and sepsis, which seem to be the most common syndromes reported, H. alvei is also associated with respiratory diseases and mixed hospital infections in humans. Since the gastrointestinal and respiratory tracts represent very common habitats for hafniae, most cases of H. alvei bacteremia originate there. H. alvei sepsis is also a serious clinical problem in the animal production industry, as infections of H. alvei can be severe, causing septicemia in commercial laying hens, pullets, and rainbow trout (19).Our knowledge of the pathogenicity of H. alvei is limited. LPS is the major virulence factor in cases of H. alvei septicemia and bacteremia (19). Studies of other virulence factors of H. alvei have reported only on the iron-scavenging mechanism, mannose-sensitive/mannose-resistant hemagglutinins, and plasmids encoding bacteriocins and alveicins (19).Most of the elucidated structures of H. alvei LPS are smooth-type molecules built up of O-specific polysaccharide, core oligosaccharide (OS), and lipid A. The O antigens of H. alvei are subdivided into 40 O serotypes (2, 28, 42). The structures of the O-specific polysaccharides from 30 serologically different H. alvei strains have been elucidated (15, 24, 26, 28, 42).So far, four types of core OS have been identified for H. alvei LPSs (9, 17, 25, 27, 30, 43). The most common core OS, isolated by mild acidic hydrolysis from LPSs of smooth H. alvei strains, is a hexasaccharide composed of two d-Glc, three l,d-Hep, and one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residues. Two l,d-Hep residues are substituted by phosphoethanolamine (PEtn) and phosphoryl (P) groups (9, 17, 25). In LPSs isolated from H. alvei PCM 1185 and 1204, core OSs are terminated with d-Galp instead of d-Glcp (27). LPSs of H. alvei containing nontypical core OSs, identical with those found in LPSs of Escherichia coli R4 (strains 23 and 1222) and Salmonella enterica Ra (strain 39), were also identified (43).The chemical structures of H. alvei O-specific chains and core OSs were elucidated using fractions obtained by mild acid hydrolysis of LPS. The procedure was optimized for the delipidation of LPS, exploiting the susceptibility of a ketosidic linkage between the inner core and lipid A to acid. However, other acid-labile linkages within the LPS could also be affected, leading to partial degradation of the isolated molecules.The presence of Kdo-containing OSs among fractions obtained by mild acid hydrolysis of LPSs, other than previously identified core OSs, makes the structural analysis of entire H. alvei LPSs difficult. Two types of trisaccharides were previously identified: (i) l-α-d-Hepp-(1→4)-[α-d-Galp-(1→7)]-α-Kdop (l-α-d-Hep is α-l-glycero-d-manno-heptose) for strains 2, 1211, 32, and 1192 (16, 23) and (ii) α-d-Galp-(1→2)-l-α-d-Hepp-(1→4)-α-Kdop for strains 1188 and 1196 (22). These Kdo-containing motifs were never located in any of the LPS segments. Thus, it is of interest to complete the structure of H. alvei LPSs with the locations of such acid-labile motifs in the structures of LPSs isolated from these bacteria.We now report on structural studies of de-N,O-acylated LPS of H. alvei 32 containing a carbohydrate backbone of lipid A, core OS, an additional trisaccharide in the outer region of the core OS, and all of the acid-labile substituents. Additionally, data obtained previously for a trisaccharide isolated from H. alvei 32 LPS (16) was complemented with detailed ¹H and ¹³C nuclear magnetic resonance (NMR) analyses and the assignment of all proton and carbon signals. Screening for the presence of these acid-labile trisaccharides, identical with those found in the strain 32 LPS, was performed on 37 different O serotypes of H. alvei LPSs with antibodies against the conjugate of the de-N,O-acylated H. alvei 32 endotoxin fragment with bovine serum albumin (BSA), specific for the isolated trisaccharide.(Part of this work was presented at the 21st International Carbohydrate Symposium, Cairns, Australia, 7 to 12 July 2002, and the 3rd German-Polish-Russian Meeting on Bacterial Carbohydrates, Wroclaw, Poland, 6 to 9 October 2004.)     

Biofilm Formation and Quorum-Sensing-Molecule Production by Clinical Isolates of Serratia liquefaciens

Serratia spp. are opportunistic human pathogens responsible for an increasing number of nosocomial infections. However, little is known about the virulence factors and regulatory circuits that may enhance the establishment and long-term survival of Serratia liquefaciens in the hospital environment. In this study, two reporter strains, Chromobacterium violaceum CV026 and VIR24, and high-resolution triple-quadrupole liquid chromatography–mass spectrometry (LC-MS) were used to detect and to quantify N-acyl-homoserine lactone (AHL) quorum-sensing signals in 20 S. liquefaciens strains isolated from clinical samples. Only four of the strains produced sufficient amounts of AHLs to activate the sensors. Investigation of two of the positive strains by high-performance liquid chromatography (HPLC)-MS confirmed the presence of significant amounts of short-acyl-chain AHLs (N-butyryl-l-homoserine lactone [C₄-HSL] and N-hexanoyl-l-homoserine lactone [C₆-HSL]) in both strains, which exhibited a complex and strain-specific signal profile that included minor amounts of other short-acyl-chain AHLs (N-octanoyl-l-homoserine lactone [C₈-HSL] and N-3-oxohexanoyl-l-homoserine lactone [OC₆-HSL]) and long-acyl-chain (C₁₀, C₁₂, and C₁₄) AHLs. No correlation between biofilm formation and the production of large amounts of AHLs could be established. Fimbria-like structures were observed by transmission electron microscopy, and the presence of the type 1 fimbrial adhesin gene fimH in all strains was confirmed by PCR. The ability of S. liquefaciens to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment, increasing the probability of causing nosocomial infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.