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The gene encoding OprF, a major outer membrane protein in Pseudomonas species, was believed to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. A novel putative extracytoplasmic function (ECF) sigma factor gene, sigX, located immediately upstream of oprF was found in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3. It was observed that SigX is involved in the regulation of other genes in P. aeruginosa.

A sequence analysis utilizing the bacterial strains and growth conditions of Escherichia coli was conducted to pFAJ26000, a small replicon of a complete nucleotide sequence 5936 bp. Replication of genes in pFAJ2600 showed that deduced proteins RepA and RepB are related to similarly organized genes in a number of cryptic plasmids from various actino-mycetes. The plasmid was also known to carry encoding putative member of the plasmid multimer resolution protein. Furthermore, the sequence analysis of pFAJ2600 showed that some of its regions are unnecessary for stable maintenance in Rhodococcus.

A study was conducted on the three copies of the element IS1415 found in Rhodococcus erythropolis NI86/21. These copies are members of the IS21 family isolated from an actinomycte and adjacent to one of which is a 47-bp sequence very similar to the conserved 5' end of integrons. Results show that the IS1415 with a chloramphenicol resistance gene is accurately transposed after delivery from a suicide vector to Rhodococcus erythropolis SQ1.

The gene encoding the ThcF protein was cloned and sequenced from Rhodococcus erythropolis NI86/21 to determine the mechanisms that mediate the biodegradation of thiocarbamates. The thcF gene encoding the thiocarbamate-inducible protein F exhibited high GC content with a molecular mass of 29,664 kDa. The ThcF protein of the noncardioform Rhodococcus erythropolis also exhibited a protein sequence that was homologous to nonheme haloperoxidase.

Analysis of the N-terminal sequences of two EPTC (S-ethyl dipropylcarbamothioate)-activated proteins from Rhodococcus sp. strain NI86 21 using two-dimensional electrophoresis helps localize the NAD+-dependent dehydrogenase and cytochrome P-450 structural genes on two specific DNA fragments. Hybridization experiments reveal that the aldehyde dehydrogenase gene is present in all strains of Rhodococcus genus while the cytochrome P-450 system is restricted to the Rhodococcus sp. strain NI86 21. Complementation experiments using the acceptor strains of EPTC-negative Rhodococcus erythropolis SQ 1 and mutant FAJ2027 confirm the participation of cytochrome P-450 system in herbicide biosafening and EPTC degradation.

N-terminal sequence analysis of Pseudomonas fluorescens reveals a surface exposed site for the proline-rich region of major outer membrane protein OprF and regions in carboxy half that are exposed but less accessible. The proline-rich domain is the most conserved region of the different P. fluorescens OprFs. Carboxy half of the OprF contains a sequence motif well conserved in enterobacterial OmpA protein.

The cloning and sequencing of the recA gene from Pseudomonas fluorescens OE 28.3 were described. DNA sequence analysis showed that the coding region of the P. fluorescens gene, specifying a protein of 352 amino acids, was preceded by an SOS box. The deduced amino acid sequence is highly similar to the RecA proteinsfrom Pseudomonas aeruginosa and Azotobacter vinelandii. In addition, a recA mutant was constructed and characterized. This mutant exhibited an increased sensitivity to UV irradiation and methyl methanesulphonate, and was severely impaired in performing homologous recombination.

Abstract Background Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. Results Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. Conclusions In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.

Abstract Background Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2. Results Differential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-βla and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of β-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis. Conclusion 2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the S. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in S. Typhimurium.

A gene cluster is identified that has encoded the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the probiotic strain Lactobacillus rhamnosus GG. The characterization of the welE mutant has shown that deprivation of the long, galactose-rich EPS molecules has resulted in an increased adherence and biofilm formation capacity of Lactobacillus rhamnosus GG, because of less shielding of adhesins such as fimbria-like structures.

Research has been conducted on Pseudomonas sp. strain BW11M1 isolate which produces antibacterial compound active against P. putida GR12-2R3. Results demonstrate that this compound is putidacin, and the cloning and characterization of the gene encoding this putidacin are described.

Research has been conducted on Streptomyces coelicolor A3(2) proteasome-lacking mutant. The study of the mutant's intracellular enzyme with chymotrypsin-like activity absent from the wild type is described.

Glycoproteins form an interesting class of macromolecules involved in bacterial-host interactions, but they are not yet widely explored in Gram-positive and beneficial species. Here, an integrated and widely applicable approach was followed to identify putative bacterial glycoproteins, combining proteome fractionation with 2D protein and glycostained gels and lectin blots. This approach was validated for the microbiota isolate Lactobacillus rhamnosus GG. The approach resulted in a list of putative glycosylated proteins receiving a 'glycosylation score'. Ultimately, we could identify 41 unique glycosylated proteins in L. rhamnosus GG (6 top-confidence, 10 high-confidence and 25 putative hits; classification based on glycosylation score). Most glycoproteins are associated with the cell wall and membrane. Identified glycoproteins include proteins involved in transport, translation, and sugar metabolism processes. A robust screening resulted in a comprehensive mapping of glycoproteins in L. rhamnosus GG. Our results reflect the glycosylation of sugar metabolism enzymes, transporters, and other proteins crucial for cell physiology. We hypothesize that protein glycosylation can confer an extra level of regulation, for example by affecting enzyme functions. This is the first systematic study of the glycoproteome of a probiotic and beneficial gut isolate. [ABSTRACT FROM AUTHOR]

Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin-based detection of glycoproteins in L actobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A ( ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin-associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin-based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used. [ABSTRACT FROM AUTHOR]

In this paper, a low-cost and low-leakage gate-first high- \(k\) metal-gate CMOS integration compatible with the high thermal budget used in a 2× node dynamic random access memory process flow is reported. The metal inserted polysilicon stack is based on HfO2 coupled with Al2O3 capping for pMOS devices, and with a TiN/Mg/TiN stack together with As ion implantation for nMOS. It is demonstrated that n and pMOS performance of 400 and 200 \(\mu \) A/ \(\mu \) m can be obtained for an OFF-state current of \(10^{-10}\) A/ \(\mu \) m, while maintaining gate and junction leakages compatible with low-power applications. Reliability and matching properties are aligned with logic gate-stacks, and the proposed solution is outperforming the La-cap-based solutions in terms of thermal stability. [ABSTRACT FROM AUTHOR]

Purified thiocarbamate-inducible ThcF of Rhodococcus erythropolis NI86/21, overexpressed in Escherichia coli, displayed several characteristics of the HASH family of enzymes that groups prokaryotic proteins of the α/β hydrolase superfamily possessing serine-dependent hydrolase and/or haloperoxidase activity. Kinetic analysis of bromination and ester hydrolysis revealed a low affinity of ThcF for model substrates. Sulfoxidation of thiocarbamates was demonstrated but probably represents a side activity due to peroxoacid generation by the enzyme. The thcF-linked thcG gene, encoding a LAL-type regulator, triggers expression of thcF in Rhodococcus. The tandem gene organization thcG-thcF is conserved in the thiocarbamate-degrading strain Rhodococcus sp. B30. It is proposed that HASH enzymes may be involved in the metabolism of plant-derived compounds. [Copyright &y& Elsevier]

Reports on the topology of the major outer membrane protein (OprF) from plant root colonizing Pseudomonas flourescens. Protease treatment of membrane-embedded OprF; Strain-dependent differences in isoelectric point of OprFs; PCR amplification and sequencing of oprF genes; Homology of OprF with OmpA-related proteins.

Background: Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2., Results: Differential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-betala and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of beta-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis., Conclusion: 2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the S. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in S. Typhimurium.

Purified thiocarbamate-inducible ThcF of Rhodococcus erythropolis NI86/21, overexpressed in Escherichia coli, displayed several characteristics of the HASH family of enzymes that groups prokaryotic proteins of the alpha/beta hydrolase superfamily possessing serine-dependent hydrolase and/or haloperoxidase activity. Kinetic analysis of bromination and ester hydrolysis revealed a low affinity of ThcF for model substrates. Sulfoxidation of thiocarbamates was demonstrated but probably represents a side activity due to peroxoacid generation by the enzyme. The thcF-linked thcG gene, encoding a LAL-type regulator, triggers expression of thcF in Rhodococcus. The tandem gene organization thcG-thcF is conserved in the thiocarbamate-degrading strain Rhodococcus sp. B30. It is proposed that HASH enzymes may be involved in the metabolism of plant-derived compounds.