Protealysin is not Secreted Constitutively.

BACKGROUND: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published. OBJECTIVE: Here, the protealysin cellular localization was studied for the first time using immunochemical methods. METHODS AND RESULTS: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm. CONCLUSION: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis.

Bioelectrochemical treatment of acid mine drainage (AMD) from an abandoned coal mine under aerobic condition.

In this study, a bioelectrochemical system (BES) was used to treat acid mine drainage (AMD) from an abandoned coal mine in the cathode chamber under aerobic condition. Activated sludge from a local wastewater treatment plant was used in the anode chamber of the BES to supply electrons to the treatment. After 7days, the pH of the cathode solution enhanced from 2.5 to 7.3. More than 99% of Al, Fe and Pb were removed, and removal rates of 93%, 91%, 89% and 69% were achieved for Cd, Zn, Mn and Co respectively with biocathode. Energy-dispersive X-ray spectroscopy (EDS) study revealed the deposition of the various types of metals on the cathode surface, and some metals were detected in the precipitates of the cathode chamber. The bacteria for AMD treatment was identified to be Serratia spp. using 16s rRNA gene amplification and sequencing. Scanning electron microscopy showed attached growth of the bacteria on the cathode. The bioelectrochemical treatment of the AMD was also compared with the biological treatment in a continuously stirred batch reactor (CSBR).

[ENTRY OF FACULTATIVE PATHOGEN SERRATIA GRIMESII INTO HELA CELLS. ELECTRON MICROSCOPIC ANALYSIS].

Facultative pathogens Serratia grimesii are able to invade eukaryotic cells where they have been found in vacuoles and free in the cytoplasm (Efremova et al., 2001; Bozhokina et al., 2011). However, efficiency of this invasion is low, and the mechanisms of the invasion related to the initial steps of the process are not known. In the present study, we have increased the invasion efficiency by incubation of HeLa cells with N-acetylcysteine (NAC) preceding the infection. In the NAC-pretreated cells, two modes of S. grimesii to enter HeLa cells were observed. In the most cases, the penetration of S. grimesii into the cell was consistent with the "zipper mechanism", involving specific interaction of bacterial invasin with a host cell surface receptor. However, in some cases, bacteria were trapped by membrane ruffling probably produced by injected bacterial proteins that trigger the bacterial uptake process, as described in the "trigger mechanism". Further elucidation of bacterial and cellular factors involved in the bacteria-host cell interaction should clarify whether two different mechanisms or a predominant one operate during S. grimesii invasion.

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens, Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces.

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48-72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N21 days of Serratia to be ca 3 Log10(CFU cm(-2)) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.

Effects of Au/Fe and Fe nanoparticles on Serratia bacterial growth and production of biosurfactant.

The overall objective of this study was to compare the effects of Au/Fe and Fe nanoparticles on the growth and performance of Serratia Jl0300. The nanoparticle effect was quantified not only by the bacterial growth on agar plate after 1 hour interaction with the nanoparticles, but also by its production of a biosurfactant from used vegetable oil. The nanoparticles were prepared using the foam method. The concentrations of the nanoparticles used for the bacterial interaction study were varied from 1 mg/L to 1 g/L. The test results showed that the effect of nanoparticles on the bacterial growth and biosurfactant production varied with nanoparticle type, concentrations, and interaction time with the bacteria. Au/Fe nanoparticles didn't show toxicity to Serratia after short time (1 h) exposure, while during 8 days fermentation Au/Fe nanoparticles inhibited the growth of Serratia as well as the biosurfactant production when the concentration of the nanoparticles was higher than 10mg/L. Fe nanoparticles showed inhibition effects to bacterial growth both after short time and long time interaction with Serratia, as well as to biosurfactant production when its concentration was higher than 100 mg/L. Based on the trends observed in this study, analytical models have been developed to predict the bacterial growth and biosurfactant production with varying concentrations of nanoparticles.

Biofouling and biodeterioration in materials stored at the Historical Archive of the Museum of La Plata, Argentine and at the National Archive of the Republic of Cuba.

The aims of this paper were to study the biofouling and biodeterioration of photos and maps stored at Historical Archive of the Museum of La Plata (HAMP), Argentine, and two repositories of the National Archive of Cuba Republic (NARC) and to carry out the physiological characterization of the isolated fungi and bacteria. The role of the environmental microbiota in the biofouling formation was also studied. Microbial assemblages in the air were sampled by sedimentation technique while those on documents were sampled by swabbering. Biofilm formation and biofouling were monitored by scanning electron microscope (SEM). Large microbial assemblages were found at NARC archives with the prevalence of genera Aspergillus, Cladosporium and Penicillium, whereas at HAMP these values were lower, Penicillium was the only fungal genus detected. Most of the fungi degraded cellulose and produced pigments and acids, and all of the isolated bacteria had proteolytic and/or cellulolytic activity. In all cases, a higher concentration of viable bacteria than of fungi was isolated from documents. These results correlated with bacterial values detected in air at NARC repositories. However, this correlation cannot be observed at HAMP where Aspergillus, Penicillium and Talaromyces helicus (teleomorph of Penicillium) were isolated. It is the first time that the last genus is reported in documents.

Serratia sp. ZF03: an efficient radium biosorbent isolated from hot-spring waters in high background radiation areas.

The aim of this study is to isolate and characterize (226)Ra biosorbing indigenous bacterial strains from soils and hot-springs containing high concentrations of (226)Ra by using biochemical and molecular approaches. Fifteen bacteria were isolated and their phylogenetic affiliations were determined based on their 16S rRNA gene and the two most relevant hypervariable regions of this gene; V3 and V6 analysis. A pigmented Serratia sp. ZF03 strain isolated from the water with (226)Ra content of 50471 mBq l(-1), caused 70% removal of (226)Ra at a radioactivity level of 50 Bq ml(-1), after 5 min and 75-80% in equilibrium time of 1 h, depending on the particular biosorption system and experimental conditions studied. The biosorption equilibrium was described by Langmuir and Freundlich isotherm models. Kinetic studies indicated that the biosorption follows pseudo-second-order kinetics. Effect of different physico-chemical parameters on (226)Ra sorption, FTIR, SEM and TEM analysis were also investigated.

Probing for actinase activity of protealysin.

The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1 : 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.

Polyhydroxybutyrate accumulation by a Serratia sp.

A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbon-limitation in continuous culture. About 1.3 mmol citrate were consumed per 450 mg biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry wt. The inclusion bodies were stained with Sudan Black and Nile Red (NR), suggesting a lipid material, which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by GC and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with NR fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increasing the overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.

Characterization of the chromosomal aac(6')-Ic gene from Serratia marcescens.

The DNA sequence of the chromosomal aac(6')-Ic gene from Serratia marcescens, which had been previously cloned (H. M. Champion, P. M. Bennett, D. A. Lewis, and D. S. Reeves, J. Antimicrob. Chemother. 22:587-596, 1988) was determined. High-pressure liquid chromatographic analysis of extracts prepared from Escherichia coli carrying the chromosomal aac(6')-Ic gene on a plasmid confirmed the presence of 6'-N-acetyltransferase activity in this strain, which was suggested by the aminoglycoside resistance profile. DNA sequence analysis of the cloned 2,057-bp PstI fragment revealed several regions of homology to previously characterized sequences from GenBank, including the rpoD and tRNA-2 genes of E. coli. Subcloning experiments confirmed the coding sequence of the aac(6')-Ic gene to be at positions 1554 to 1992. The predicted amino acid sequence of the AAC(6')-Ic protein suggested that it was the third member of a family of AAC(6') proteins which included a coding region identified between the aadB and aadA genes of Tn4000 and an AAC(6') protein encoded by pUO490, which was isolated from Enterobacter cloacae. Primer extension analysis suggested that the -35 region of the aac(6')-Ic promoter overlapped a large palindromic sequence which may be involved in the regulation of the aac(6')-Ic gene. Hybridization experiments utilizing a restriction fragment from the aac(6')-Ic gene showed that all S. marcescens organisms carried this gene whether or not the AAC(6')-I resistance profile was expressed. Organisms other than Serratia spp. did not hybridize to this probe.

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.

Multiple fimbrial haemagglutinins in Serratia species.

Fifty-six strains from nine Serratia species, grown under a variety of cultural conditions, were examined in rocked-tile tests for the presence of haemagglutinins (HAs) and with the electron microscope for fimbriae. All strains were haemagglutinating; most (71%) formed two or three of the different kinds of HA detected. These were: (i) a mannose-sensitive HA (MS-HA); (ii) a mannose-resistant, klebsiella-like HA (MR/K-HA); (iii) two mannose-resistant, proteus-like HAs (MR/P-HA), one of which showed broad-spectrum HA activity against different species of erythrocytes and the other narrow-spectrum HA activity. Five types of fimbriae associated with the different HAs were observed. This description of the fimbrial HAs in nine species of Serratia is the most comprehensive so far produced and should provide a basis for future studies directed towards elucidating the ecological role of the adhesins in in-vivo colonization by serratiae as commensals or pathogens.

New fimbrial hemagglutinin in Serratia species.

Strains of Serratia marcescens, Serratia liquefaciens, Serratia marinorubra, and Serratia plymuthica produced one or more of the following hemagglutinins (HAs): mannose-sensitive HA and mannose-resistant K-HA (MR/K-HA) and P-HA (MR/P-HA) (J. P. Duguid and D. C. Old, in E. H. Beachey (ed.), Bacterial adherence, vol. 6., p. 185-217, 1980). Most strains (82%) were multiply hemagglutinating. The properties of the three HAs are described. Each HA was associated with a distinct type of fimbria: mannose-sensitive HA with type 1 fimbriae. MR/K-HA with type 3 fimbriae, and MR/P-HA with a new type of thin fimbriae provisionally called MR/P fimbriae. This is the first report of the production of MR/P-HA and MR/P fimbriae by Serratia species. The range of Serratia HAs, which may reflect in vivo colonization potential, is more complex than previously reported.