scholarly journals The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 923-935 ◽  
Author(s):  
Nicole Hansmeier ◽  
Andreas Albersmeier ◽  
Andreas Tauch ◽  
Thomas Damberg ◽  
Robert Ros ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Affinity Purification ◽  
Regulatory Protein ◽  
Direct Repeat ◽  
Peptide Mass Fingerprinting ◽  
Gene Encoding ◽  
Force Microscopy ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Rapid Amplification

The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

scholarly journals Identification and Phenotypic Characterization of Sphingomonas wittichii Strain RW1 by Peptide Mass Fingerprinting Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

2005 ◽  
Vol 71 (5) ◽  
pp. 2442-2451 ◽  
Author(s):  
Rolf U. Halden ◽  
David R. Colquhoun ◽  
Eric S. Wisniewski
Keyword(s):  
Mass Spectrometry ◽  
Laser Desorption ◽  
Peptide Mass Fingerprinting ◽  
Phenotypic Characterization ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Sphingomonas Wittichii

ABSTRACT Mass spectrometry is a potentially attractive means of monitoring the survival and efficacy of bioaugmentation agents, such as the dioxin-mineralizing bacterium Sphingomonas wittichii strain RW1. The biotransformation activity of RW1 phenotypes is determined primarily by the presence and concentration of the dioxin dioxygenase, an enzyme initiating the degradation of both dibenzo-p-dioxin and dibenzofuran (DF). We explored the possibility of identifying and characterizing putative cultures of RW1 by peptide mass fingerprinting (PMF) targeting this characteristic phenotypic biomarker. The proteome from cells of RW1—grown on various media in the presence and absence of DF—was partially purified, tryptically digested, and analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mascot online database queries allowed statistically significant identification of RW1 in disrupted, digested cells (P < 0.01 to 0.05) and in digested whole-cell extracts (P < 0.00001 to 0.05) containing hundreds of proteins, as determined by two-dimensional gel electrophoresis. Up to 14 peptide ions of the alpha subunit of the dioxin dioxygenase (43% protein coverage) were detected in individual samples. A minimum of 107 DF-grown cells was required to identify dioxin degradation-enabled phenotypes. The technique hinges on the detection of multiple characteristic peptides of a biomarker that can reveal at once the identity and phenotypic properties of the microbial host expressing the protein. The results demonstrate the power of PMF of minimally processed microbial cultures as a sensitive and specific technique for the positive identification and phenotypic characterization of certain microorganisms used in biotechnology and bioremediation.

scholarly journals Identification of Polypeptides Expressed in Response to Vinyl Chloride, Ethene, and Epoxyethane in Nocardioides sp. Strain JS614 by Using Peptide Mass Fingerprinting

2007 ◽  
Vol 73 (13) ◽  
pp. 4368-4372 ◽  
Author(s):  
Adina S. Chuang ◽  
Timothy E. Mattes
Keyword(s):  
Vinyl Chloride ◽  
Laser Desorption ◽  
Peptide Mass Fingerprinting ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

ABSTRACT Enzymes expressed in response to vinyl chloride, ethene, and epoxyethane by Nocardioides sp. strain JS614 were identified by using a peptide mass fingerprinting (PMF) approach. PMF provided insight concerning vinyl chloride biodegradation in strain JS614 and extends the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry as a tool to enhance characterization of biodegradation pathways.

scholarly journals Functional Genomic Analysis of Global Regulator NolR in Sinorhizobium meliloti

2005 ◽  
Vol 18 (12) ◽  
pp. 1340-1352 ◽  
Author(s):  
Hancai Chen ◽  
Ke Gao ◽  
Eva Kondorosi ◽  
Adam Kondorosi ◽  
Barry G. Rolfe
Keyword(s):  
Sinorhizobium Meliloti ◽  
Regulatory Protein ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Nucleotide Metabolism ◽  
Conjugative Transfer ◽  
Environmental Stimuli ◽  
Peptide Mass ◽  
Functional Genomic Analysis ◽  
Associated Proteins

NolR is a regulator of nodulation genes present in species belonging to the genera Rhizobium and Sinorhizobium. The expression of the nolR gene in Sinorhizobium meliloti AK631 was investigated in relation to stage of growth, availability of nutrients, and different environmental stimuli using the nolR::lacZ fusion report system. It has been shown that the nolR gene is regulated in a population-density-dependent fashion and influenced by a number of environmental stimuli, including nutrients, pH, and oxygen. Exploration of the physiological functions of NolR under various laboratory conditions has shown that NolR is required for the optimal growth of the bacteria on solid media, optimal survival of the bacteria in carbon-starved minimal medium, and after heat shock challenge. NolR also is involved in recipient-induced conjugative transfer of a plasmid. Proteome analysis of strain AK631 and its Tn5-induced nolR-deficient mutant EK698 revealed that a functional NolR induced significant differences in the accumulation of 20 polypeptides in peptide mass fingerprinting early-log-phase cultures and 48 polypeptides in stationary-phase cultures. NolR acted mainly as a repressor in the early-log-phase cultures, whereas it acted as both repressor and activator in the stationaryphase cultures. The NolR protein and 59 NolR-associated proteins have been identified by peptide mass fingerprinting. The NolR protein was differentially expressed only in the NolR+ wild-type strain AK631 but not in its NolR- derivative EK698, confirming that no functional NolR was produced in the mutant. The NolR-associated proteins have diverse functions in amino acid metabolism, carbohydrate metabolism, lipid metabolism, nucleotide metabolism, energy metabolism, metabolism of Co-factors, and cellular adaptation and transportation. These results further support our previous proposal that the NolR is a global regulatory protein which is required for the optimization of nodulation, bacterial growth and survival, and conjugative transfer of a plasmid.

scholarly journals Molecular Characterization of Radial Spoke Subcomplex Containing Radial Spoke Protein 3 and Heat Shock Protein 40 in Sperm Flagella of the AscidianCiona intestinalis

2005 ◽  
Vol 16 (2) ◽  
pp. 626-636 ◽  
Author(s):  
Yuhkoh Satouh ◽  
Potturi Padma ◽  
Toshifusa Toda ◽  
Nori Satoh ◽  
Hiroyuki Ide ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Sequence Similarity ◽  
Protein A ◽  
Gel Filtration ◽  
Peptide Mass Fingerprinting ◽  
Distal Portion ◽  
Radial Spoke ◽  
Flight Mass Spectrometry ◽  
Peptide Mass

Members of the heat-shock protein (HSP)40 regulate the protein folding activity of HSP70 proteins and help the functional specialization of this molecular chaperone system in various types of cellular events. We have recently identified Hsp40 as a component of flagellar axoneme in the ascidian Ciona intestinalis, suggesting a correlation between Hsp40 related chaperone system and flagellar function. In this study, we have found that Ciona 37-kDa Hsp40 is extracted from KCl-treated axonemes with 0.5 M KI solution and comigrates with radial spoke protein (RSP)3 along with several proteins as a complex through gel filtration and ion exchange columns. Peptide mass fingerprinting with matrix-assisted laser desorption ionization/time of flight/mass spectrometry revealed that other proteins in the complex include a homolog of sea urchin spokehead protein (homolog of RSP4/6), a membrane occupation and recognition nexus repeat protein with sequence similarity with meichroacidin, and a functionally unknown 33-kDa protein. A spoke head protein, LRR37, is not included in the complex, suggesting that the complex constructs the stalk of radial spoke. Immunoelectron microscopy indicates that Hsp40 is localized in the distal portion of spoke stalk, possibly at the junction between spoke head and the stalk.

scholarly journals Comparative Analysis of Extracellular and Intracellular Proteomes of Listeria monocytogenes Strains Reveals a Correlation between Protein Expression and Serovar

2008 ◽  
Vol 74 (23) ◽  
pp. 7399-7409 ◽  
Author(s):  
Emilie Dumas ◽  
Bruno Meunier ◽  
Jean-Louis Berdagué ◽  
Christophe Chambon ◽  
Mickaël Desvaux ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Peptide Mass Fingerprinting ◽  
Etiologic Agent ◽  
Extracellular Proteins ◽  
Health Concern ◽  
Two Dimensional Gel Electrophoresis ◽  
Extracellular Proteome ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Phylogenetic Lineages

ABSTRACT Listeria monocytogenes, the etiologic agent of listeriosis, remains a serious public health concern, with its frequent occurrence in food environments coupled with a high mortality rate. Among the 13 serovars, human listeriosis is mostly associated with the serovar 4b, 1/2b, and 1/2a strains. To investigate the diversity of L. monocytogenes, the intracellular and extracellular proteins of 12 strains were analyzed by two-dimensional gel electrophoresis. These strains had different origins, belonged to different serovars (4b, 1/2a, and 1/2b), and presented with different levels of virulence in chicken embryos. The clustering of the strains in two groups based on proteomic patterns is in agreement with the L. monocytogenes phylogenetic lineages. Statistical analysis did not allow for identification of proteins specific to the isolate origin or the virulence level of the strains, but 26 and 21 protein spots were shown to be significantly overexpressed and underexpressed, respectively, in the six strains of serovar 1/2a (lineage II) compared to strains of serovar 1/2b or 4b. Moreover, a penicillin-binding protein was specific for serovar 1/2b and two protein spots identified as a serine protease were specific to serovar 4b. These protein spots, identified through peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry, were essentially found in the extracellular proteome and may have uses as potential markers for serotyping and risk analysis.

scholarly journals Heat Shock Proteome Analysis of Wild-Type Corynebacterium glutamicum ATCC 13032 and a Spontaneous Mutant Lacking GroEL1, a Dispensable Chaperone

2005 ◽  
Vol 187 (3) ◽  
pp. 884-889 ◽  
Author(s):  
Carlos Barreiro ◽  
Eva González-Lavado ◽  
Sven Brand ◽  
Andreas Tauch ◽  
Juan F. Martín
Keyword(s):  
Heat Shock ◽  
Corynebacterium Glutamicum ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Start Codon ◽  
Northern Analysis ◽  
Spontaneous Mutant ◽  
Wild Type ◽  
Peptide Mass ◽  
Heat Response

ABSTRACT Proteome analysis of Corynebacterium glutamicum ATCC 13032 showed that levels of several proteins increased drastically in response to heat shock. These proteins were identified as DnaK, GroEL1, GroEL2, ClpB, GrpE, and PoxB, and their heat response was in agreement with previous transcriptomic results. A major heat-induced protein was absent in the proteome of strain 13032B of C. glutamicum, used for genome sequencing in Germany, compared with the wild-type ATCC 13032 strain. The missing protein was identified as GroEL1 by matrix-assisted laser desorption ionization-time of flight peptide mass fingerprinting, and the mutation was found to be due to an insertion sequence, IsCg1, that was integrated at position 327 downstream of the translation start codon of the groEL1 gene, resulting in a truncated transcript of this gene, as shown by Northern analysis. The GroEL1 chaperone is, therefore, dispensable in C. glutamicum. On the other hand, GroEL2 appears to be essential for growth. Based on these results, the role of the duplicate groEL1 and groEL2 genes is analyzed.

Proteomic Changes Induced by Podophyllotoxin in Human Cervical Carcinoma HeLa Cells

2013 ◽  
Vol 41 (01) ◽  
pp. 163-175 ◽  
Author(s):  
Bochan Wang ◽  
Lifeng Chen ◽  
Hong Zhen ◽  
Li Zhou ◽  
Ping Shi ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cervical Carcinoma ◽  
Hela Cells ◽  
Cell Communication ◽  
Peptide Mass Fingerprinting ◽  
Human Cervical Carcinoma ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Associated Proteins ◽  
Growth Of Tumor

Podophyllotoxin, a kind of lignan extracted from the Podophyllum plant, has been shown to inhibit the growth of various carcinoma cells. However, the molecular mechanism remains unclear. In this study, the inhibition of cell growth and changes in protein expression induced by podophyllotoxin were investigated in human cervical carcinoma HeLa cells. Our results demonstrate that Podophyllotoxin inhibits HeLa cell growth and induces apoptosis. By using proteomic techniques, seven proteins were found to be significantly regulated by podophyllotoxin compared to the untreated control; among them, four were down-regulated and three were up-regulated. All of the seven proteins were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after in-gel trypsin digestion. Five of these proteins are involved in protein metabolism, and the other two play roles in cell communication and signaling transduction pathways. It is suggested that the effect of podophyllotoxin on the growth of tumor cells is significantly related to the metabolism-associated proteins.

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