Biology subjects

Mera, P. E.

Publications and source records attributed to Mera, P. E..

7 recordsLinked to original sources

bioRxiv2026

A dual-function regulatory element couples ParB expression and DNA substrate specificity

Chromosome segregation is essential for cell survival. Most bacteria encode the chromosome partitioning ParABS system. Although even small changes in ParA or ParB levels disrupt genome maintenance, the mechanisms that control their abundance have remained unresolved. Using Caulobacter crescentus, we provide the first mechanistic evidence that ParB levels are regulated post-transcriptionally. Through single-nucleotide substitutions and compensatory mutation analyses, we identify an mRNA secondary structure at the 5' end of the parB transcript that enhances ParB cellular abundance. Loss of this regulatory mechanism sensitizes cells to modest increases in ParA levels, causing cell death. Additionally, we demonstrate that ParB substrate specificity is not determined solely by the central helix-turn-helix domain that interacts with parS but is unexpectedly modulated by the N-terminal domain. Together, our findings reveal an uncharacterized layer of chromosome segregation control and highlight post-transcriptional regulation of ParB abundance as a potential mechanism for maintaining the precise ParA-ParB balance required for bacterial viability.

microbiology↗

bioRxiv2025

Cell size regulation in bacteria: a tale of old regulators with new mechanisms

Successful proliferation of bacteria requires the coordination of multiple cellular events. The precise coordination between the replication of the chromosome with cell growth and division is essential for daughter cells to inherit an intact copy of the genome with the appropriate cell size. However, the molecular mechanisms that temporally and spatially integrate these essential processes have remained elusive in the field. Using the model system C. crescentus, we uncovered a previously unrecognized role for the conserved chromosome replication initiator DnaA in regulating cell growth and division dynamics. Through mutant analyses, transcriptional profiling, biochemical and high-resolution imaging, our data demonstrate that DnaA modulates cell size through direct transcriptional activation of enzymes involved in cell wall biosynthesis. We identify a conserved cell wall biosynthetic enzyme, MurD, as a key downstream effector and demonstrate its unexpected ability to restrict cell size. Both in vivo and in vitro analyses revealed that the transcriptional regulation of murD requires the ATP-bound form of DnaA, the same nucleotide-bound form required for initiating chromosome replication. These findings highlight how bacteria leverage the conserved nucleotide-dependent switch, DnaA-ATP, to temporally orchestrate spatially distinct processes ensuring accurate progression of the cell cycle.

microbiology↗

bioRxiv2023

TipN involvement with centromere segregation in Caulobacter crescentus

Bacterias ability to maintain chromosomal integrity throughout their life cycle is crucial for their survival. In Caulobacter crescentus, the polar factor TipN has been proposed to be involved with the partitioning system ParABS. However, cells with tipN knocked out display subtle parS segregation defects. We hypothesized that TipNs role with parS segregation is obscured by other forces that are ParABS-independent. To test our hypothesis, we removed one of those forces - chromosome replication - and analyzed the role of TipN with ParA. We first demonstrate that ParA retains its ability to transport the centromeric region parS from the stalked pole to the opposite pole in the absence of chromosome replication. Our data revealed that in the absence of chromosome replication, TipN becomes essential for ParAs ability to transport parS. Furthermore, we identify a potential connection between the replication initiator DnaA and TipN. Although TipN is not essential for viability, tipN knockout cells lose viability when the regulation of DnaA levels is altered. Our data suggest that the DnaA-dependent susceptibility of tipN knockout cells is connected to parS segregation. Collectively, this work provides insights into the complex regulation involved in the coordination of chromosome replication and segregation in bacteria.

microbiology↗

bioRxiv2023

To let go or not to let go: how ParA can impact the release of the chromosomal anchoring in Caulobacter crescentus

Chromosomal maintenance is vital for the survival of bacteria. In Caulobacter crescentus, chromosome replication initiates at ori and segregation is delayed until the nearby centromere-like region parS is replicated. Our understanding of how this sequence of events is regulated remains limited. The segregation of parS has been shown to involve multiple steps including polar release from anchoring protein PopZ, slow movement, and fast ParA-dependent movement to opposite cell pole. In this study, we demonstrate that ParAs competing attractions from PopZ and from DNA are critical for segregation of parS. Interfering with this balance of attractions - by expressing a variant ParA-R195E unable to bind DNA and thus favoring interactions exclusively between ParA-PopZ - results in cell death. Our data revealed that ParA-R195Es sole interactions with PopZ obstruct PopZs ability to release the polar anchoring of parS resulting in cells with multiple parS loci fixed at one cell pole. We show that the inability to separate and segregate multiple parS loci from the pole is specifically dependent on the interaction between ParA and PopZ. Interfering with interactions between PopZ and the partitioning protein ParB, which is the interaction that anchors parS at the cell pole, does not rescue the ability of cells to separate the fixed parS loci when expressing parA-R195E. Thus, ParA and PopZ appear to have a distinct conversation from ParB yet can impact the release of ParB-parS from the anchoring at the cell pole. Collectively, our results reveal that the initial steps in chromosome segregation are highly regulated.

microbiology↗

bioRxiv2022

ParA and its functions that go beyond chromosome segregation in Caulobacter crescentus

Maintaining the integrity of the chromosome after the completion of each cell cycle is paramount for bacterial survival. Mechanistic details remain incomplete for how bacteria manage to retain intact chromosomes in each daughter cell after each cell division. In this study, we examined the partitioning protein ParA and its functions on chromosomal maintenance that go beyond triggering the onset of chromosome segregation. Our data demonstrate that ParA can promote the onset of chromosome replication initiation in Caulobacter crescentus cells with sub-physiological levels of the replication initiator protein DnaA. Increasing the cellular levels of ParA results in over-initiation of chromosome replication in this bacterium. We show that the ability of ParA to impact replication initiation is independent from ParAs ability to trigger the onset of chromosome segregation. Surprisingly, our work revealed that perturbing the balance of the components of ParAs nucleotide-dependent cycle can have severe defects in cell cycle coordination and can potentially be lethal to the cell. Increasing the levels of different forms of ParA also impacted cell length independent of their replication initiation frequencies. Our results, together with past observations, suggest a model where ParA can serve as a checkpoint coordinating various cell cycle events involved in maintenance of the chromosome.

microbiology↗

bioRxiv2022

Class II UvrA protein Ecm16 requires ATPase activity to render resistance against echinomycin

II.Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the Nucleotide Excision Repair (NER) protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 preferentially binds double-stranded DNA over single-stranded DNA and is likely to primarily interact with the backbone of DNA using a nucleotide-independent binding mode. Ecm16s binding affinity for DNA increased significantly when the DNA is intercalated with echinomycin. Ecm16 can repair echinomycin-induced DNA damage independently of NER. Like UvrA, Ecm16 has ATPase activity and this activity is essential for Ecm16s ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each others function. Increasing the cellular levels of UvrA in E. coli was insufficient to render echinomycin resistance. Similarly, Ecm16 was unable to repair DNA damage that is specific to UvrA. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin. Our results, together with past observations, suggest a model where Ecm16 recognizes double helix distortions caused by echinomycin and repairs the problem independently of NER.

microbiology↗

bioRxiv2019

Chromosome dynamics in bacteria: triggering replication at opposite location and segregation in opposite direction

The accurate onset of chromosome replication and segregation are fundamental for the survival of the cell. In bacteria, regulation of chromosome replication lies primarily at the initiation step. The bacterial replication initiator DnaA recognizes the origin of replication (ori) and opens this double stranded site allowing for the assembly of the DNA replication machinery. Following the onset of replication initiation, the partitioning protein ParA triggers the onset of chromosome segregation by direct interactions with ParB-bound to the centromere. The subcellular organization of ori and centromere are maintained after the completion of each cell cycle. It remains unclear what triggers the onset of these key chromosome regulators DnaA and ParA. One potential scenario is that the microenvironment of where the onset of replication and segregation take place hosts the regulators that trigger the activity of DnaA and ParA. In order to address this, we analyzed whether the activity of DnaA and ParA are restricted to only one site within the cell. In non-dividing cells of the alpha proteobacterium Caulobacter crescentus, ori and centromere are found near the stalked pole. To test DnaAs ability to initiate replication away from the stalked pole, we engineered a strain where movement of ori was induced in the absence of chromosome replication. Our data show that DnaA can initiate replication of the chromosome independently of the subcellular localization of ori. Furthermore, we discovered that the partitioning protein ParA was functional and could segregate the replicated centromere in the opposite direction from the new pole toward the stalked pole. We showed that the organization of the ParA gradient can be completely reconstructed in the opposite orientation by rearranging the location of the centromere. Our data reveal the high flexibility of the machineries that trigger the onset of chromosome replication and segregation in bacteria. Our work also provides insights into the coordination between replication and segregation with the cellular organization of specific chromosomal loci.

microbiology↗