Absolute quantification of microbial proteomes at different states by directed mass spectrometry

Schmidt, Alexander; Beck, Martin; Malmström, Johan; Lam, Henry; Claassen, Manfred; Campbell, D. S.; Aebersold, Ruedi

doi:10.1038/msb.2011.37

Cited by 97 publications

(119 citation statements)

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“…interrogans encounters diverse environmental signals at different stages of its life cycle. Studies of individual genes and whole transcriptomes and proteomes have revealed a number of L. interrogans genes that are differentially expressed when the spirochete is incubated under conditions likely to be encountered during host infection (3)(4)(5)(6)(7)(8)(9)(10)(11). Levels of specific outer membrane proteins also differ during acute infection of the liver (12) and in leptospires exiting the host in urine (13).…”

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confidence: 99%

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

2013

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fThe spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5= untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in doublestranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA fMet -mRNA ternary complex was inhibited unless a 5= deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5= UTR and the first six codons were inserted between the Escherichia coli L-arabinose promoter and bgaB (␤-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the ␤-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5= UTR slightly diminished expression at each temperature tested. Finally, the expression level of ␤-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5= UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.

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confidence: 99%

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

2013

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“…This profile of increased expression in such specific DNA repair genes combined with decreased cell growth and motility is not seen in the response to physiologic osmolarity, serum, DMCs or various antibiotics (25,28,68,78). However, it has a similar profile to the response to ciprofloxacin, a DNA gyrase inhibitor indicated for infections caused by Gram-negative bacteria (68,79). In L. interrogans as well in P. aeruginosa, it causes up-regulation of recA concomitant with increase in DNA repair, prophages and mutagenesis, in addition to decreased cell growth, motility, virulence and ciprofloxacinspecific repair genes (recG, ruvABC) (68,80,81).…”

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

“…In other situations, some components seem to be individually controlled. For example, LIC12603 (pectinesterase) increases expression after treatment with serum (68). The pore-forming toxin LIC10172 is strongly expressed in blood, liver and kidney of infected hamsters, while LIC12611 (pblA-like)…”

Section: Discussionmentioning

confidence: 99%

“…However, it has a similar profile to the response to ciprofloxacin, a DNA gyrase inhibitor indicated for infections caused by Gram-negative bacteria (68,79). In L. interrogans as well in P. aeruginosa, it causes up-regulation of recA concomitant with increase in DNA repair, prophages and mutagenesis, in addition to decreased cell growth, motility, virulence and ciprofloxacinspecific repair genes (recG, ruvABC) (68,80,81).…”

Section: Discussionmentioning

confidence: 99%

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Caracterization of the SOS response in Leptospira interrogans sorovar Copenhageni

Fonseca¹

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ACKNOWLEDGMENTS / AGRADECIMENTOSAgradeço aos vários mentores que tive ao longo desses 5 anos: ao Paulo Lee Ho, pela orientação e por me dar a liberdade necessária para desenvolver o projeto e deixá-lo com a minha cara; à Renata Costa, minha coorientadora de coração, pela amizade e empolgação científica compartilhada; ao Fredy Gueiros, por acompanhar meu doutorado e me ajudar mesmo sem ter nenhuma obrigação. Ao Alan Grossman, por abrir as portas do seu laboratório e me receber como uma das suas, sendo um verdadeiro mentor e exemplo de cientista.A todas as pessoas que fazem do Grossman's lab um lugar que dá vontade de voltar: Janet (pelos scripts e por não me deixar congelar no inverno), C. Lee (pela competência, paciência e vídeos de gatinho), Charlotte, Laurel e Monica (pela amizade e festas do brigadeiro).O Instituto Butantan é puro amor, e conheci muitas pessoas incríveis lá. Aos pesquisadores do Lab de Biotecnologia Molecular I (Jô, Enéas, Léo, Eliane, Alessandra), agradeço não só pelo apoio científico, mas pelos papos-cabeça, ensinamentos e por aguentarem meu mau-humor enquanto escrevia essa tese. Às técnicas Nídia e Aline, pois sem elas eu não encontraria nem minhas pipetas na bancada. A todas as amigas que fiz, Leptospira is a basal genus in an ancient group of bacteria, the spirochetes. The pathogenic species are responsible for leptospirosis, a disease with worldwide distribution and of public health importance in developed tropical countries. L. interrogans serovar Copenhageni is the agent for the majority of human leptospirosis in Brazil. In this work, we used a great variety of experimental approaches to characterize the SOS system in this serovar, to identify its impact in general DNA damage response, as well as to assess the DNA repair toolbox owned by pathogenic and saprophytic leptospires. We identified an additional repressor LexA, acquired by lateral gene transfer, exclusively in serovar Copenhageni. We also observed that UV-C irradiation led to massive death of cells and blockage of cell division in the survivors. Both repressors were active and we identified the sequences responsible for binding to promoters. However, the LexA1 SOS box was redefined after a de novo motif search on LexA1 ChIP-seq enriched sequences. This regulator was able to bind to at least 25 loci in the genome. DNA damage also caused a massive rearrangement of metabolism: increase in expression was observed in transposon and prophage genes, in addition to DNA repair pathways and mutagenesis inducers; on the other hand, motility, general metabolism and almost all virulence genes were repressed. Two induced prophages provided several proteins with useful functions. We also assessed the DNA repair-related genes presented by the three species of Leptospira: the saprophytic L.

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What is the total number of protein molecules per cell volume? A call to rethink some published values

2013

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Novel methods such as mass-spectrometry enable a view of the proteomes of cells in unprecedented detail. Recently, these efforts have culminated in quantitative measurements of the number of copies per cell for most expressed proteins in organisms ranging from bacteria to mammalian cells. Here, we estimate the expected total number of proteins per unit of cell volume using known parameters related to the composition of cells such as the fraction of cell mass that is protein, and the average protein length. Using simple arguments, we estimate a range of 2–4 million proteins per cubic micron (i.e. 1 fL) in bacteria, yeast, and mammalian cells. Interestingly, we find that measured values that are reported for fission yeast and mammalian cells are often about 3–10 times lower. We discuss this apparent discrepancy and how to use the estimate as benchmark to recalibrate proteome-wide quantitative censuses or to revisit assumptions about cell composition.We estimate the expected total number of proteins per unit cell volume as 2–4 million proteins per cubic micron. Some reported values for fission yeast and mammalian cells using mass spectrometry are 3–10 times lower than these estimates. We discuss this apparent discrepancy and how to recalibrate proteome-wide quantitative censuses.

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Absolute quantification of microbial proteomes at different states by directed mass spectrometry

Cited by 97 publications

References 63 publications

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

Role for cis -Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

Caracterization of the SOS response in Leptospira interrogans sorovar Copenhageni

What is the total number of protein molecules per cell volume? A call to rethink some published values

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