Functional Genomic Analysis of Global Regulator NolR in Sinorhizobium meliloti

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.

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

Journal of Bacteriology ◽

10.1128/jb.185.17.5029-5036.2003 ◽

2003 ◽

Vol 185 (17) ◽

pp. 5029-5036 ◽

Cited By ~ 49

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.

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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 ◽

10.1099/mic.0.28673-0 ◽

2006 ◽

Vol 152 (4) ◽

pp. 923-935 ◽

Cited By ~ 37

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.

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

Journal of Bacteriology ◽

10.1128/jb.184.13.3485-3491.2002 ◽

2002 ◽

Vol 184 (13) ◽

pp. 3485-3491 ◽

Cited By ~ 129

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.

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Effects of AiiA-Mediated Quorum Quenching in Sinorhizobium meliloti on Quorum-Sensing Signals, Proteome Patterns, and Symbiotic Interactions

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-7-0843 ◽

2007 ◽

Vol 20 (7) ◽

pp. 843-856 ◽

Cited By ~ 27

Author(s):

Mengsheng Gao ◽

Hancai Chen ◽

Anatol Eberhard ◽

Matthew R. Gronquist ◽

Jayne B. Robinson ◽

...

Keyword(s):

Quorum Sensing ◽

Sinorhizobium Meliloti ◽

Structural Similarity ◽

Peptide Mass Fingerprinting ◽

Quorum Quenching ◽

Homoserine Lactone ◽

Protein Accumulation ◽

In Planta ◽

Symbiotic Interactions ◽

Peptide Mass

Many behaviors in bacteria, including behaviors important to pathogenic and symbiotic interactions with eukaryotic hosts, are regulated by a mechanism called quorum sensing (QS). A “quorum-quenching” approach was used here to identify QS-regulated behaviors in the N-fixing bacterial symbiont Sinorhizobium meliloti. The AiiA lactonase from Bacillus produced in S. meliloti was shown to enzymatically inactivate S. meliloti's N-acyl homoserine lactone (AHL) QS signals, thereby disrupting normal QS regulation. Sixty proteins were differentially accumulated in the AiiA-producing strain versus the control in early log or early stationary phase cultures. Fifty-two of these QS-regulated proteins, with putative functions that include cell division, protein processing and translation, metabolite transport, oxidative stress, and amino acid metabolism, were identified by peptide mass fingerprinting. Transcription of representative genes was reduced significantly in the AiiA-producing strain, although the effects of AiiA on protein accumulation did not always correspond to effects on transcription. The QS signal-deficient strain was reduced significantly in nodule initiation during the first 12 h after inoculation onto Medicago truncatula host plants. The AiiA lactonase also was found to substantially inactivate two of the AHL mimic compounds secreted by M. truncatula. This suggests some structural similarity between bacterial AHLs and these mimic compounds. It also indicates that quorum quenching could be useful in identifying Sinorhizobium genes that are affected by such host QS mimics in planta.

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Matrix-assisted laser desorption–ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures

Journal of Chromatography B Biomedical Sciences and Applications ◽

10.1016/s0378-4347(00)00446-1 ◽

2001 ◽

Vol 752 (2) ◽

pp. 311-322 ◽

Cited By ~ 32

Author(s):

Stephanie Lamer ◽

Peter R. Jungblut

Keyword(s):

Mass Spectrometry ◽

Proteome Analysis ◽

Laser Desorption ◽

Peptide Mass Fingerprinting ◽

Ionization Mass ◽

Laser Desorption Ionization ◽

Desorption Ionization ◽

Peptide Mass ◽

Matrix Assisted Laser ◽

Matrix Assisted Laser Desorption

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

Canadian Journal of Plant Science ◽

10.1139/cjps-2018-0115 ◽

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.

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Proteome Analysis of Differentially Displayed Proteins As a Tool for the Investigation of Symbiosis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.9.995 ◽

2000 ◽

Vol 13 (9) ◽

pp. 995-1009 ◽

Cited By ~ 116

Author(s):

Siria H. A. Natera ◽

Nelson Guerreiro ◽

Michael A. Djordjevic

Keyword(s):

Sinorhizobium Meliloti ◽

Cultured Cells ◽

Proteome Analysis ◽

Sequence Information ◽

Down Regulation ◽

Symbiotic Interaction ◽

Two Dimensional Gel Electrophoresis ◽

Nitrogen Acquisition ◽

Flight Mass Spectrometry ◽

Peptide Mass

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.

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

Journal of Bacteriology ◽

10.1128/jb.187.3.884-889.2005 ◽

2005 ◽

Vol 187 (3) ◽

pp. 884-889 ◽

Cited By ~ 37

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.

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