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Transcriptomic and phenotypic characterization of a Bacillus subtilis strain without extracytoplasmic function ? factors.

ABSTRACT: Bacillus subtilis encodes seven extracytoplasmic function (ECF) ? factors. Three (?(M), ?(W), and ?(X)) mediate responses to cell envelope-active antibiotics. The functions of ?(V), ?(Y), ?(Z), and ?(YlaC) remain largely unknown, and strong inducers of these ? factors and their regulons have yet to be defined. Here, we define transcriptomic and phenotypic differences under nonstress conditions between a strain carrying deletions in all seven ECF ? factor genes (the ?7ECF mutant), a ?MWX triple mutant, and the parental 168 strain. Our results identify >80 genes as at least partially dependent on ECF ? factors, and as expected, most of these are dependent on ?(M), ?(W), or ?(X), which are active at a significant basal level during growth. Several genes, including the eps operon encoding enzymes for exopolysaccharide (EPS) production, were decreased in expression in the ?7ECF mutant but affected less in the ?MWX mutant. Consistent with this observation, the ?7ECF mutant (but not the ?MWX mutant) showed reduced biofilm formation. Extending previous observations, we also note that the ?MWX mutant is sensitive to a variety of antibiotics and the ?7ECF mutant is either as sensitive as, or slightly more sensitive than, the ?MWX strain to these stressors. These findings emphasize the overlapping nature of the seven ECF ? factor regulons in B. subtilis, confirm that three of these (?(M), ?(W), and ?(X)) play the dominant role in conferring intrinsic resistance to antibiotics, and provide initial insights into the roles of the remaining ECF ? factors.

SUBMITTER: Luo Y

PROVIDER: S-EPMC2953670 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Transcriptomic and phenotypic characterization of a Bacillus subtilis strain without extracytoplasmic function σ factors.

Luo Yun Y Asai Kei K Sadaie Yoshito Y Helmann John D JD

Journal of bacteriology 20100903 21

Bacillus subtilis encodes seven extracytoplasmic function (ECF) σ factors. Three (σ(M), σ(W), and σ(X)) mediate responses to cell envelope-active antibiotics. The functions of σ(V), σ(Y), σ(Z), and σ(YlaC) remain largely unknown, and strong inducers of these σ factors and their regulons have yet to be defined. Here, we define transcriptomic and phenotypic differences under nonstress conditions between a strain carrying deletions in all seven ECF σ factor genes (the Δ7ECF mutant), a ΔMWX triple m ...[more]

PMID: 20817771

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Project description:Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF ?s) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like ?s (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF ?s. We discovered new targets for individual ?s, confirmed others by overexpressing individual ECF ?s, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF ? gene and its demonstrated or putative anti-? gene and also a strain in which all 11 ECF ? and anti-? genes were deleted. This all-ECF ? deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF ?s were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF ? and anti-? genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [?s]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.

Project description:Extracytoplasmic function σ factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-σ factor. Mycobacterium tuberculosis membrane-associated anti-σ factors have a small, stable RNA gene A (ssrA)-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and a substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-σ factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (X-Ala-Ala) leads to an order-of-magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-σ interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-σ factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-σ degradation, thereby regulating the release of free σ from the σ/anti-σ complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF σ factors.IMPORTANCE The ability of Mycobacterium tuberculosis to quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) σ factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the σ factor from the anti-σ is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-σ factors have an ssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX-mediated proteolysis and affects the cellular abundance of ECF σ factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.

Dataset Information

Transcriptomic and phenotypic characterization of a Bacillus subtilis strain without extracytoplasmic function ? factors.

Publications

Transcriptomic and phenotypic characterization of a Bacillus subtilis strain without extracytoplasmic function σ factors.

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