Identification of iron-responsive genes in Proteus vulgaris.

Iron is essential for almost all bacteria, and iron homeostasis is precisely controlled by the ferric uptake regulator (Fur). The Fur regulons have been well characterized in some model bacteria, yet little is known in the common opportunistic pathogen Proteus vulgaris. In this study, Fur regulon and iron-responsive genes in P. vulgaris were mainly defined by in silico and proteomic analyses. The results showed that about 250 potential Fur-regulated operons including 14 transcriptional factors were predicted, while 559 proteins exhibited differential expression in response to iron deficiency, not all being directly regulated by Fur, such as transcriptional factors lexA, recA, narL, and arcA. Collectively, these results demonstrated that Fur functioned as a global regulatory protein to repress or activate expression of a large repertoire of genes in P. vulgaris; besides, not all the iron-responsive genes were directly regulated by Fur, whereas indirectly regulated through other mechanisms such as additional transcriptional regulatory proteins.

Comparative genomic identification and characterization of npcRNA homologs in Proteus vulgaris.

Proteus vulgaris is a rod-shaped Gram-negative bacterium known to be the member of Enterobacteriaceae that is able to cause disease in human being. Generally, non-protein-coding RNAs (npcRNAs) do not code for proteins, but they play a vital role in gene regulation at the RNA level including pathogenicity. The present study aims at elucidating homologous npcRNAs from other bacteria in Proteus vulgaris. A comparative genomic analysis was carried out to identify npcRNA homolog of other Enterobacteriaceae pathogens in Proteus vulgaris. A total of 231 npcRNAs previously reported in Salmonella typhi, Salmonella typhimurium and Escherichia coli were screened using BLASTn tool against Proteus vulgaris genome. Interestingly, 33 npcRNAs are homologs to Proteus vulgaris. Northern blot analysis of 6 out of 33 npcRNA candidates confirmed their expression and showed that most of them are differentially expressed during lag, exponential and stationary growth phases. This study is the first approach of identification and characterization of npcRNAs in P. vulgaris. Hence, this could be a pioneer study to further validate the regulatory functions of these npcRNAs to fill the gaps in understanding of the pathogenicity of P. vulgaris.

A Novel Non-Coding RNA CsiR Regulates the Ciprofloxacin Resistance in Proteus vulgaris by Interacting with emrB mRNA.

Bacterial non-coding RNAs (ncRNAs) play important regulatory roles in various physiological metabolic pathways. In this study, a novel ncRNA CsiR (ciprofloxacin stress-induced ncRNA) involved in the regulation of ciprofloxacin resistance in the foodborne multidrug-resistant Proteus vulgaris (P. vulgaris) strain P3M was identified. The survival rate of the CsiR-deficient strain was higher than that of the wild-type strain P3M under the ciprofloxacin treatment condition, indicating that CsiR played a negative regulatory role, and its target gene emrB was identified through further target prediction, quantitative real-time PCR (qRT-PCR), and microscale thermophoresis (MST). Further studies showed that the interaction between CsiR and emrB mRNA affected the stability of the latter at the post-transcriptional level to a large degree, and ultimately affected the ciprofloxacin resistance of P3M. Notably, the base-pairing sites between CsiR and emrB mRNAs were highly conserved in other sequenced P. vulgaris strains, suggesting that this regulatory mechanism may be ubiquitous in this species. To the best of our knowledge, this is the first identification of a novel ncRNA involved in the regulation of ciprofloxacin resistance in P. vulgaris species, which lays a solid foundation for comprehensively expounding the antibiotic resistance mechanism of P. vulgaris.

Bacterial Biotransformation and Anticancer Activities of Betulin against A549, HepG2 and 5RP7 Cancer Cell Lines.

BACKGROUND: A pentacyclic lupenane-type natural triterpenoid, betulin, has attracted attention in the field of medicinal chemistry since it exhibited a variety of biological activities, including anticancer activity. OBJECTIVE: The aim of this present work was to obtain derivatives of betulin through bacterial biotransformation and investigate its anticancer activity against A549, HepG2 and 5RP7 cancer cell lines. METHODS: Bacterial biotransformation studies were continued in an MBH broth medium for 7 days at 35oC. Anticancer activities of betulin against A549, HepG2 and 5RP7 cell lines were carried out using XTT assay, and their selectivity was determined using a healthy cell line of NIH/3T3. Cell proliferation ELISA, BRDU (colorimetric) assay was used for measuring proliferation in replicative cells in which DNA synthesis occurs. Flow cytometric analysis was used for measuring apoptotic cell percentages, caspase 3 activation and mitochondrial membrane potential. RESULTS: Bacterial biotransformation studies with 7 bacteria of Staphylococcus aureus ATCC 6538, Proteus vulgaris NRRL B-123, Bacillus subtilis NRRL B-4378, Streptomyces griseolus NRRL B-1062, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 43300 and Bacillus velezensis NRRL B-14580 produced no metabolite. In in vitro anticancer activity studies, betulin was found to exert anticancer activity against A549, HepG2 and 5RP7 cell lines with IC50 values of 207.7, 125.0 and 28.3 µg/mL, whereas SI values were found to be 30, 50 and 223, respectively. Early and late apoptotic percentages of betulin were found as 9.6, 12.1 and 85.4% on A549, HepG2 and 5RP7, respectively, while caspase 3 positive cell percentages were 2.3, 28.7 and 13.3% for IC50 concentrations. In addition, betulin caused G1 cell cycle arrest (49.5%) on 5RP7 cell line. CONCLUSION: The results have been shown that betulin activities against A549 and HepG2 cell lines were nonselective and limited its cytotoxic activity against healthy cells, but it is possible to say that it exerted selective activity against 5RP7 cell (28.33±1.53 µg/mL). Betulin effects on apoptosis were found to be dosedependent, while its effect on caspase 3 activation, mitochondrial membrane potential, and cell cycle arrest on G0/G1 phase was not dependent on doses. Therefore, betulin could be a good candidate for the treatment of H-ras active cancer types.

Proteus vulgaris - Pt electrode system for urea to nitrogen conversion in synthetic urine.

One of the most challenging problems when trying to recycle urine for different purposes is the removal of urea. In this project we studied an ureolysis system using the bacterium Proteus vulgaris for the transformation of urea to ammonia and its subsequent oxidation to nitrogen at a Pt working electrode. Our system was tested under different pH, microbial reaction times, and urea and bacteria concentrations. Our results indicate that a pH8 is optimal for the combined Proteus vulgaris urease activity and the ammonia oxidation reaction at a Pt electrode. The reaction time and concentration dependence on the ammonia oxidation reaction current densities was also studied. Results showed limited ammonia oxidation under high urea concentrations in ~2.5×109cfu/mL Proteus vulgaris in synthetic urine.

Inactivation of the arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of proteus vulgaris.

Colistin has become a last-line antibiotic for the treatment of multidrug-resistant bacterial infections; however, resistance to colistin has emerged in recent years. Some bacteria, such as Proteus and Serratia spp., are intrinsically resistant to colistin although the exact mechanism of resistance is unknown. Here we identified the molecular support for intrinsic colistin resistance in Proteus spp. by comparative genomic, transcriptomic and proteomic analyses of colistin-susceptible (CSUR P1868_S) and colistin-resistant (CSUR P1867_R) strains of an atypical Proteus vulgaris. A significant difference in outer membrane glycoside structures in both strains that was corroborated by MALDI-TOF/MS analysis was found, which showed an absence of 4-amino-4-deoxy-l-arabinose (L-Ara4N) in the outer membrane lipid A moiety of the susceptible strain. Comparative genomic analysis with other resistant strains of P. vulgaris available in a local database found a mutation in the arnBCADTEF operon of the susceptible strain. Transcriptomic analysis of genes belonging to the arnBCADTEF operon showed a significant decrease in mRNA expression level of these genes in the susceptible strain, supporting addition of L-Ara4N in the outer membrane lipid A moiety as an explanation for colistin resistance. Insertion of the arnD gene that was suggested to be altered in the susceptible strain by in silico analysis led to a 16-fold increase of colistin MIC in the susceptible strain, confirming its role in colistin resistance in this species. Here we show that constitutive activation of the arn operon and addition of L-Ara4N is the main molecular mechanism of colistin resistance in P. vulgaris.

Understanding the adhesion mechanism of a mucin binding domain from Lactobacillus fermentum and its role in enteropathogen exclusion.

Lactobacillus species possesses surface exposed Mucin Binding Protein (MucBP) which plays a role in adhesion to gastrointestinal mucin. MucBP contains one or more mucin binding domain (MBD), the functionality of which has yet not been characterized thoroughly. Here, we have characterized a 93-amino acid MBD (MBD93) of MucBP (LAF_0673) from Lactobacillus fermentum. Multiple sequence alignment of L. fermentum MBD93 exhibited ∼60% sequence homology with MBDs from other Lactobacillus species. Further, we cloned, expressed and purified MBD93 from Escherichia coli as N-terminal histidine-tagged protein (6X His-MBD93). The purified MBD93 was able to bind to mucin and showed strong affinity towards the terminally expressed mucin glycans viz. N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), Galactose (Gal), and Sialic acid (N-acetylneuraminic acid; Neu5Ac). In silico experiments further confirmed the interaction between homology modeled MBD93 to mucin glycans through hydrogen-bonding with its surface amino acid residues Ser57, Pro58, Ile60, Tyr63 and Ala65. We also have demonstrated that MBD93 was able to inhibit the adhesion of enteric pathogens, including E. coli, Salmonella Paratyphi A, Shigella sonnei and Proteus vulgaris to mucin. Our results suggested that L. fermentum MBD93 is a functionally sufficient unit to act as an adhesin and to protect from invading enteric pathogens.

Production of Omega-3 Fatty Acid Ethyl Esters from Menhaden Oil Using Proteus vulgaris Lipase-Mediated One-Step Transesterification and Urea Complexation.

An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (ω-3 PUFA EEs). First, the lyophilized recombinant lipase K80 (LyoK80) was used to perform the transesterification reaction of menhaden oil and ethanol. LyoK80 produced the ω-3 PUFA EEs with a conversion yield of 82 % in the presence of 20 % water content via a three-step ethanol-feeding process; however, in a non-aqueous condition, LyoK80 produced only a slight amount of the ω-3 PUFA EEs. To enhance its reaction properties, the lipase K80 was immobilized on a hydrophobic bead to derive ImmK80; the biochemical properties and substrate specificity of ImmK80 are similar to those of LyoK80. ImmK80 was then used to produce ω-3 PUFA EEs in accordance with the same transesterification reaction. Unlike LyoK80, ImmK80 achieved a high ω-3 PUFA EE conversion yield of 86 % under a non-aqueous system via a one-step ethanol-feeding reaction. The ω-3 PUFA EEs were purified up to 92 % using a urea complexation method.

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation.

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.

One-step biosynthesis of α-ketoisocaproate from L-leucine by an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase from Proteus vulgaris.

This work aimed to develop a whole-cell biotransformation process for the production of α-ketoisocaproate from L-leucine. A recombinant Escherichia coli strain was constructed by expressing an L-amino acid deaminase from Proteus vulgaris. To enhance α-ketoisocaproate production, the reaction conditions were optimized as follows: whole-cell biocatalyst 0.8 g/L, leucine concentration 13.1 g/L, temperature 35 °C, pH 7.5, and reaction time 20 h. Under the above conditions, the α-ketoisocaproate titer reached 12.7 g/L with a leucine conversion rate of 97.8%. In addition, different leucine feeding strategies were examined to increase the α-ketoisocaproate titer. When 13.1 g/L leucine was added at 2-h intervals (from 0 to 22 h, 12 addition times), the α-ketoisocaproate titer reached 69.1 g/L, while the leucine conversion rate decreased to 50.3%. We have developed an effective process for the biotechnological production of α-ketoisocaproate that is more environmentally friendly than the traditional petrochemical synthesis approach.

Production of acylated homoserine lactone by a novel marine strain of Proteus vulgaris and inhibition of its swarming by phytochemicals.

A marine strain of Proteus vulgaris capable of activating multiple acylated homoserine lactone (AHL)-based reporter cultures was isolated. The cognate signal molecule was characterized as octanoyl homoserine lactone (OHL) and its production was observed to be growth dependent, with maximum production (5.675 µg l(-1)) at 24 h growth. The strain exhibited swarming, but its motility was not affected upon addition of pure OHL or culture supernatant. Phytochemicals such as quercitin and berberine chloride inhibited OHL production and reduced swarming. FliA, the predominantly upregulated protein during swarming, was considered as a possible target for these inhibitors, and docking of the two most active and two least active inhibitors to this protein suggested preferential binding of the former set of compounds. Apart from adding new evidence to AHL production in Proteus vulgaris, active inhibitors shortlisted from this study could help in identifying lead compounds to act against this opportunistic pathogen of the respiratory and gastrointestinal tract.

Screening of phenylpyruvic acid producers and optimization of culture conditions in bench scale bioreactors.

Alpha keto acids are deaminated forms of amino acids that have received significant attention as feed and food additives in the agriculture and medical industries. To date, their production has been commonly performed at shake-flask scale with low product concentrations. In this study, production of phenylpyruvic acid (PPA), which is the alpha keto acid of phenylalanine was investigated. First, various microorganisms were screened to select the most efficient producer. Thereafter, growth parameters (temperature, pH, and aeration) were optimized in bench scale bioreactors to maximize both PPA and biomass concentration in bench scale bioreactors, using response surface methodology. Among the four different microorganisms evaluated, Proteus vulgaris was the most productive strain for PPA production. Optimum temperature, pH, and aeration conditions were determined as 34.5 °C, 5.12, and 0.5 vvm for PPA production, whereas 36.9 °C, pH 6.87, and 0.96 vvm for the biomass production. Under these optimum conditions, PPA concentration was enhanced to 1,054 mg/L, which was almost three times higher than shake-flask fermentation concentrations. Moreover, P. vulgaris biomass was produced at 3.25 g/L under optimum conditions. Overall, this study demonstrated that optimization of growth parameters improved PPA production in 1-L working volume bench-scale bioreactors compared to previous studies in the literature and was a first step to scale up the production to industrial production.

Structure of the Proteus vulgaris HigB-(HigA)2-HigB toxin-antitoxin complex.

Bacterial toxin-antitoxin (TA) systems regulate key cellular processes to promote cell survival during periods of stress. During steady-state cell growth, antitoxins typically interact with their cognate toxins to inhibit activity presumably by preventing substrate recognition. We solved two x-ray crystal structures of the Proteus vulgaris tetrameric HigB-(HigA)2-HigB TA complex and found that, unlike most other TA systems, the antitoxin HigA makes minimal interactions with toxin HigB. HigB adopts a RelE family tertiary fold containing a highly conserved concave surface where we predict its active site is located. HigA does not cover the solvent-exposed HigB active site, suggesting that, in general, toxin inhibition is not solely mediated by active site hindrance by its antitoxin. Each HigA monomer contains a helix-turn-helix motif that binds to its own DNA operator to repress transcription during normal cellular growth. This is distinct from antitoxins belonging to other superfamilies that typically only form DNA-binding motifs upon dimerization. We further show that disruption of the HigB-(HigA)2-HigB tetramer to a HigBA heterodimer ablates operator binding. Taken together, our biochemical and structural studies elucidate the novel molecular details of the HigBA TA system.

Relationship between chemical structure and biological activity of alkali metal o-, m- and p-anisates. FT-IR and microbiological studies.

In this work we investigated relationship between molecular structure of alkali metal o-, m-, p-anisate molecules and their antimicrobial activity. For this purpose FT-IR spectra for lithium, sodium, potassium, rubidium and caesium anisates in solid state and solution were recorded, assigned and analysed. Microbial activity of studied compounds was tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Proteus vulgaris. In order to evaluate the dependency between chemical structure and biological activity of alkali metal anisates the statistical analysis (multidimensional regression and principal component) was performed for selected wavenumbers from FT-IR spectra and parameters that describe microbial activity of anisates. The obtained statistical equations show the existence of correlation between molecular structure of anisates and their biological properties.

Detection of volatile compounds produced by microbial growth in urine by selected ion flow tube mass spectrometry (SIFT-MS).

Selected ion flow tube-mass spectrometry has been used to measure the volatile compounds occurring in the headspace of urine samples inoculated with common urinary tract infection (UTI)-causing microbes Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterococcus faecalis, or Candida albicans. This technique has the potential to offer rapid and simple diagnosis of the causative agent of UTIs.

Tryptophan inhibits Proteus vulgaris TnaC leader peptide elongation, activating tna operon expression.

Expression of the tna operon of Escherichia coli and of Proteus vulgaris is induced by L-tryptophan. In E. coli, tryptophan action is dependent on the presence of several critical residues (underlined) in the newly synthesized TnaC leader peptide, WFNIDXXL/IXXXXP. These residues are conserved in TnaC of P. vulgaris and of other bacterial species. TnaC of P. vulgaris has one additional feature, distinguishing it from TnaC of E. coli; it contains two C-terminal lysine residues following the conserved proline residue. In the present study, we investigated L-tryptophan induction of the P. vulgaris tna operon, transferred on a plasmid into E. coli. Induction was shown to be L-tryptophan dependent; however, the range of induction was less than that observed for the E. coli tna operon. We compared the genetic organization of both operons and predicted similar folding patterns for their respective leader mRNA segments. However, additional analyses revealed that L-tryptophan action in the P. vulgaris tna operon involves inhibition of TnaC elongation, following addition of proline, rather than inhibition of leader peptide termination. Our findings also establish that the conserved residues in TnaC of P. vulgaris are essential for L-tryptophan induction, and for inhibition of peptide elongation. TnaC synthesis is thus an excellent model system for studies of regulation of both peptide termination and peptide elongation, and for studies of ribosome recognition of the features of a nascent peptide.

Enhanced decolorization and biodegradation of textile azo dye Scarlet R by using developed microbial consortium-GR.

A developed consortium-GR, consisting of Proteus vulgaris NCIM-2027 (PV) and Micrococcus glutamicus NCIM-2168 (MG), completely decolorized an azo dye Scarlet R under static anoxic condition with an average decolorization rate of 16,666 microg h(-1); which is much faster than that of the pure cultures (PV, 3571 microg h(-1); MG, 2500 microg h(-1)). Consortium-GR gave best decolorization performance with nearly complete mineralization of Scarlet R (over 90% TOC and COD reduction) within 3h, much shorter relative to the individual strains. Induction in the riboflavin reductase and NADH-DCIP reductase was observed in the consortium, suggesting the involvement of these enzymes during the fast decolorization process. The FTIR and GC-MS analysis showed that 1,4-benzenediamine was formed during decolorization/degradation of Scarlet R by consortium-GR. Phytotoxicity studies revealed no toxicity of the biodegraded products of Scarlet R by consortium-GR. In addition, consortium-GR applied for mixture of industrial dyes showed 88% decolorization under static condition with significant reduction in TOC (62%) and COD (68%) within 72 h, suggesting potential application of this microbial consortium in bioremediation of dye-containing wastewater.

Inhibition of Candida albicans isocitrate lyase activity by sesterterpene sulfates from the tropical sponge Dysidea sp.

Seven sesterterpene sulfates (1-7) were isolated from the tropical sponge Dysidea sp. and their inhibitory activities against isocitrate lyase (ICL) from Candida albicans were evaluated. Among the isolated natural products compound 6 and 7 were found to be strong ICL inhibitors. The isolated compounds (1-7) also showed potent antibacterial effect against Bacillus subtilis and Proteus vulgaris, but did not display antifungal activity.

Declination of copper toxicity in pigeon pea and soil system by growth-promoting Proteus vulgaris KNP3 strain.

The copper-resistant (1318 microM CuSO(4).5H(2)O) strain KNP3 of Proteus vulgaris was isolated from soil near the Panki power plant, Kanpur, India, and was used to inoculate pigeon pea (Cajanus cajan var. UPS-120) seeds grown in soil for 60 days in the presence of 600 microM CuSO(4).5H(2)O. A study of siderophore production (126.34 +/- 0.52 microg ml(-1)) and its subsequent effects on plant growth promotion under in situ conditions was conducted. The parameters that were monitored included the plants' wet weight, dry weight, shoot length, chlorophyll content, and concentration of copper in plant roots and shoots. The results showed that the strain caused a significant (p < 0.05) increase in wet weight, dry weight, root length, shoot growth, and chlorophyll content (57.8%, 60%, 19.7%, 47.8%, and 36.3%, respectively) in the presence of copper. Furthermore, the strain reduced accumulation of Cu in the roots and shoots to 36.8% and 60.5%, respectively. Apart from this, copper concentration in the soil was measured on 0, 7, 15, 30, and 45 days consecutively and the results indicated that the bioinoculant KNP3 causes a significant decrease in Cu concentration in soil (55.6%), which was unlikely in the control (10.5%) treatment. The data suggested that the bacterial strain has the ability to protect plants against the inhibitory effects of copper besides reducing the copper load of the soil.

Production of volatile aroma compounds by bacterial strains isolated from different surface-ripened French cheeses.

Twelve bacterial strains belonging to eight taxonomic groups: Brevibacterium linens, Microbacterium foliorum, Arthrobacter arilaitensis, Staphylococcus cohnii, Staphylococcus equorum, Brachybacterium sp., Proteus vulgaris and Psychrobacter sp., isolated from different surface-ripened French cheeses, were investigated for their abilities to generate volatile aroma compounds. Out of 104 volatile compounds, 54 volatile compounds (identified using dynamic headspace technique coupled with gas chromatography-mass spectrometry [GC-MS]) appeared to be produced by the different bacteria on a casamino acid medium. Four out of eight species used in this study: B. linens, M. foliorum, P. vulgaris and Psychrobacter sp. showed a high flavouring potential. Among these four bacterial species, P. vulgaris had the greatest capacity to produce not only the widest varieties but also the highest quantities of volatile compounds having low olfactive thresholds such as sulphur compounds. Branched aldehydes, alcohols and esters were produced in large amounts by P. vulgaris and Psychrobacter sp. showing their capacity to breakdown the branched amino acids. This investigation shows that some common but rarely mentioned bacteria present on the surface of ripened cheeses could play a major role in cheese flavour formation and could be used to produce cheese flavours.