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.

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

2013 ◽  
Vol 195 (11) ◽  
pp. 2707-2707
Author(s):  
C. Barreiro ◽  
E. Gonzalez-Lavado ◽  
S. Brand ◽  
A. Tauch ◽  
J. F. Martin
Keyword(s):  
Heat Shock ◽  
Corynebacterium Glutamicum ◽  
Proteome Analysis ◽  
Spontaneous Mutant ◽  
Wild Type

scholarly journals Proteomic Analysis of Wild-Type Sinorhizobium meliloti Responses to N-Acyl Homoserine Lactone Quorum-Sensing Signals and the Transition to Stationary Phase

2003 ◽  
Vol 185 (17) ◽  
pp. 5029-5036 ◽  
Author(s):  
Hancai Chen ◽  
Max Teplitski ◽  
Jayne B. Robinson ◽  
Barry G. Rolfe ◽  
Wolfgang D. Bauer
Keyword(s):  
Quorum Sensing ◽  
Stationary Phase ◽  
Proteomic Analysis ◽  
Sinorhizobium Meliloti ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Homoserine Lactone ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Peptide Mass

ABSTRACT Proteome analysis revealed that two long-chain N-acyl homoserine lactones (AHLs) produced by Sinorhizobium meliloti 1021 induced significant differences in the accumulation of more than 100 polypeptides in early-log-phase cultures of the wild type. Fifty-six of the corresponding proteins have been identified by peptide mass fingerprinting. The proteins affected by addition of these two AHLs had diverse functions in carbon and nitrogen metabolism, energy cycles, metabolite transport, DNA synthesis, and protein turnover. Two hours of exposure to 3-oxo-C16:1-homoserine lactone (3-oxo-C16:1-HL) affected the accumulation of 40 of the 56 identified proteins, whereas comparable exposure to C14-HL affected 13 of the 56 proteins. Levels of four proteins were affected by both AHLs. Exposure to 3-oxo-C16:1-HL for 8 h affected the accumulation of 17 proteins, 12 of which had reduced accumulation. Of the 80 proteins identified as differing in accumulation between early-log- and early-stationary-phase cultures, only 13 were affected by exposure to 3-oxo-C16:1-HL or C14-HL. These results provide a foundation for future studies of the functions regulated by AHL quorum sensing in S. meliloti and help to establish proteomic analysis as a powerful global approach to the identification of quorum-sensing regulatory patterns in wild-type bacteria.

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 Hypoxic Response of Mycobacterium tuberculosis Studied by Metabolic Labeling and Proteome Analysis of Cellular and Extracellular Proteins

2002 ◽  
Vol 184 (13) ◽  
pp. 3485-3491 ◽  
Author(s):  
Ida Rosenkrands ◽  
Richard A. Slayden ◽  
Janne Crawford ◽  
Claus Aagaard ◽  
Clifton E. Barry ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Extracellular Proteins ◽  
Metabolic Labeling ◽  
Low Oxygen ◽  
Hypoxic Response ◽  
Peptide Mass ◽  
Oxygen Conditions

ABSTRACT The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

scholarly journals Characterization of proteinase–adhesin complexes of Porphyromonas gingivalis

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2381-2394 ◽  
Author(s):  
Rishi D. Pathirana ◽  
Neil M. O'Brien-Simpson ◽  
Paul D. Veith ◽  
Peter F. Riley ◽  
Eric C. Reynolds
Keyword(s):  
Porphyromonas Gingivalis ◽  
Specific Activity ◽  
Binding Constants ◽  
Peptide Mass Fingerprinting ◽  
Host Protein ◽  
Wild Type ◽  
Equilibrium Binding ◽  
Peptide Mass ◽  
Type V ◽  
Triton X

Proteinase–adhesin complexes of Porphyromonas gingivalis wild-type and RgpA and Kgp mutants were extracted using a Triton X-114 procedure and purified using arginine-affinity chromatography. The complexes were then characterized by peptide mass fingerprinting (PMF) and their equilibrium binding constants, immunogenicity and ability to induce protection as vaccines in the murine lesion model determined. The Triton X-114 procedure resulted in consistently higher yield and specific activity of the wild-type (wt) complex compared with that produced by the previously published sonication method. PMF and N-terminal sequencing of the purified wt complex showed that it consisted of the previously identified Arg-specific proteinase RgpAcat, the Lys-specific proteinase Kgpcat and adhesin domains RgpAA1, RgpAA2, RgpAA3, KgpA1 and KgpA2. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpAA4, indicated that this band contained C-terminally truncated KgpA1 (which has an identical N-terminus to RgpAA4) as well as the HagAA1* adhesin. Analysis of the Triton X-114 extracted complexes from the P. gingivalis isogenic mutants kgp (RgpA complex) and rgpA (Kgp complex) suggested that the Kgp complex consisted of Kgpcat, KgpA1 and KgpA2/HagAA2 and that the RgpA complex consisted of RgpAcat, RgpAA1, HagAA1*, RgpAA2 and RgpAA3. Each of the complexes was found to have equilibrium binding constants (K D) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lower K D values for binding to each host protein compared with the sonication-wt complex, or the Triton-RgpA complex and Triton-Kgp complex. Furthermore, the Triton-wt complex induced a stronger antibody response to the A1 adhesins and tended to be more effective in providing protection in the mouse lesion model compared with the sonication-wt complex.

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.

Comparative proteome analysis of drought-sensitive and drought-tolerant maize leaves under osmotic stress

2019 ◽  
Vol 99 (4) ◽  
pp. 467-479
Author(s):  
Yuhe Pei ◽  
Jianfen Bai ◽  
Xinmei Guo ◽  
Meiai Zhao ◽  
Qingmei Ma ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Inbred Lines ◽  
Limiting Factor ◽  
Two Dimensional Gel Electrophoresis ◽  
Maize Production ◽  
Drought Tolerant ◽  
Maize Inbred Lines ◽  
Peptide Mass

Drought is a major yield-limiting factor in maize production. Osmotic stress was applied to two maize inbred lines with polyethylene glycol 6000 treatments. Proteins from the leaves were analyzed by two-dimensional gel electrophoresis and peptide mass fingerprinting at two time points, 24 and 48 h after osmotic stress. Thirty-five proteins were differentially expressed between control and treatment groups in the two maize inbred lines. In ‘Qi319’, a drought-tolerant inbred line, there were five up-regulated proteins at 24 h and 13 up-regulated and one down-regulated protein at 48 h. In drought-sensitive line ‘Zheng58’, 10 proteins were up-regulated at 24 h, while six proteins were up-regulated at 48 h. The 35 proteins were subjected to mass spectrometry and 17 proteins were successfully identified. These proteins were classified into six categories: photosynthesis-related, energy and metabolism, signaling pathways, protein synthesis, defense-related, and unclassified.

Transcriptional regulation of histidine biosynthesis genes in Corynebacterium glutamicum

2010 ◽  
Vol 56 (2) ◽  
pp. 178-187 ◽  
Author(s):  
Samil Jung ◽  
Jae-Yeon Chun ◽  
Sei-Heun Yim ◽  
Soo-Suk Lee ◽  
Choong-Il Cheon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Corynebacterium Glutamicum ◽  
Single Copy ◽  
Start Codon ◽  
Leader Region ◽  
Wild Type ◽  
T Box ◽  
Attenuation Mechanism ◽  
Series Systems ◽  
Cat Gene

Corynebacterium glutamicum , a gram-positive bacterium, has been widely used for industrial amino acid production. Corynebacterium glutamicum his genes are located and transcribed in two unlinked loci, hisEG and hisDCB–orf1–orf2–hisHA–impA–hisFI. The latter his operon starts the transcription at the C residue localized 196 bp upstream of the hisD ATG start codon. Our computer-based sequence analysis showed that the region corresponding to the untranslated 5′ end of the transcript, named the hisD leader region, displays the typical features of the T-box transcriptional attenuation mechanism. Therefore, expression of the cat reporter gene under the control of the wild-type or mutated hisD leader regions was tested in multi-copy (pProm and pTer series) and in single-copy (pInt series) systems under conditions of sufficient or limited histidine. Our mutational studies led to the conclusion that the CAU histidine specifier and 5′-UGGA-3′ sequence in the hisD leader region are required for the hisDCB–orf1–orf2–hisHA–impA–hisFI gene regulation. The cat gene expression from the wild-type leader region was negatively regulated by histidine. However, the cat gene expression from mutated leader regions was irresponsive to the level of histidine in the growth medium. Taken together, we propose that a T-box mediated attenuation mechanism is responsible for the gene expression of the hisDCB–orf1–orf2–hisHA–impA–hisFI operon in C. glutamicum.

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.

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