Cells attaching to the extracellular matrix spontaneously acquire front-rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front-rear polarity in spreading cells.

• MeSH
• Cell Adhesion genetics   physiology   MeSH
• Cell Line MeSH
• Fibroblasts cytology   metabolism   MeSH
• Focal Adhesion Protein-Tyrosine Kinases genetics   metabolism   MeSH
• Rats MeSH
• Microscopy, Phase-Contrast MeSH
• Cell Movement genetics   physiology   MeSH
• Cell Polarity genetics   physiology   MeSH
• Receptors for Activated C Kinase genetics   metabolism   MeSH
• RNA Interference MeSH
• Cell Shape genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, PMs1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding β and β' subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.

• MeSH
• RNA, Bacterial metabolism   MeSH
• Gene Deletion MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• RNA, Small Untranslated genetics   metabolism   MeSH
• Mycobacterium smegmatis enzymology   genetics   growth & development   metabolism   MeSH
• Gene Expression Profiling MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The σI sigma factor from Bacillus subtilis is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI Further analysis revealed that the majority of these genes were affected indirectly by σI The σI regulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σI in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism B. subtilisIMPORTANCE In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σI regulon from the industrially important model Gram-positive bacterium Bacillus subtilis We reveal that σI affects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery.

• MeSH
• Bacillus subtilis genetics   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases genetics   MeSH
• Transcription, Genetic * MeSH
• Operon MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Regulon MeSH
• Sigma Factor genetics   MeSH
• Transcriptome MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Bacillus subtilis enzymology   genetics   MeSH
• Bacterial Proteins isolation & purification   metabolism   MeSH
• DNA-Directed RNA Polymerases chemistry   isolation & purification   metabolism   MeSH
• DNA metabolism   MeSH
• Transcription Elongation, Genetic MeSH
• Phenotype MeSH
• Transcription, Genetic * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH