Dcw Gene Research Articles

Diversification of division mechanisms in endospore-forming bacteria revealed by analyses of peptidoglycan synthesis in Clostridioides difficile

The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions.

Ancient origin and constrained evolution of the division and cell wall gene cluster in Bacteria.

The division and cell wall (dcw) gene cluster in Bacteria comprises 17 genes encoding key steps in peptidoglycan synthesis and cytokinesis. To understand the origin and evolution of this cluster, we analysed its presence in over 1,000 bacterial genomes. We show that the dcw gene cluster is strikingly conserved in both gene content and gene order across all Bacteria and has undergone only a few rearrangements in some phyla, potentially linked to cell envelope specificities, but not directly to cell shape. A large concatenation of the 12 most conserved dcw cluster genes produced a robust tree of Bacteria that is largely consistent with recent phylogenies based on frequently used markers. Moreover, evolutionary divergence analyses show that the dcw gene cluster offers advantages in defining high-rank taxonomic boundaries and indicate at least two main phyla in the Candidate Phyla Radiation (CPR) matching a sharp dichotomy in dcw gene cluster arrangement. Our results place the origin of the dcw gene cluster in the Last Bacterial Common Ancestor and show that it has evolved vertically for billions of years, similar to major cellular machineries such as the ribosome. The strong phylogenetic signal, combined with conserved genomic synteny at large evolutionary distances, makes the dcw gene cluster a robust alternative set of markers to resolve the ever-growing tree of Bacteria.

The Conserved Dcw Gene Cluster of R. sphaeroides Is Preceded by an Uncommonly Extended 5' Leader Featuring the sRNA UpsM.

Cell division and cell wall synthesis mechanisms are similarly conserved among bacteria. Consequently some bacterial species have comparable sets of genes organized in the dcw (division and cell wall) gene cluster. Dcw genes, their regulation and their relative order within the cluster are outstandingly conserved among rod shaped and gram negative bacteria to ensure an efficient coordination of growth and division. A well studied representative is the dcw gene cluster of E. coli. The first promoter of the gene cluster (mraZ1p) gives rise to polycistronic transcripts containing a 38 nt long 5’ UTR followed by the first gene mraZ. Despite reported conservation we present evidence for a much longer 5’ UTR in the gram negative and rod shaped bacterium Rhodobacter sphaeroides and in the family of Rhodobacteraceae. This extended 268 nt long 5’ UTR comprises a Rho independent terminator, which in case of termination gives rise to a non-coding RNA (UpsM). This sRNA is conditionally cleaved by RNase E under stress conditions in an Hfq- and very likely target mRNA-dependent manner, implying its function in trans. These results raise the question for the regulatory function of this extended 5’ UTR. It might represent the rarely described case of a trans acting sRNA derived from a riboswitch with exclusive presence in the family of Rhodobacteraceae.

Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation

ObjectiveTo investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). MethodsFifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration–response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. ResultsAll 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 μg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 μg/mL. ConclusionsThe present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

YI.4 Gonococcal Genomics Shows Impact of Recombination on Obscuring Phylogenetic Signal and Disseminating Resistance Loci

BackgroundRecombination plays a significant role in the plasticity of the Neisseria gonorrhoeaegenome by generating antigenic diversity and as a mechanism of spread of antibiotic resistance elements. Extensive recombination in a...

The Mycobacterium tuberculosis serine/threonine kinase PknL phosphorylates Rv2175c: Mass spectrometric profiling of the activation loop phosphorylation sites and their role in the recruitment of Rv2175c

Although Mycobacterium tuberculosis (M. tb) comprises 11 serine/threonine protein kinases, the mechanisms of regulation of these kinases and the nature of their endogenous substrates remain largely unknown. Herein, we characterized the M. tb kinase PknL by demonstrating that it expresses autophosphorylation activity and phosphorylates Rv2175c. On-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, five phosphorylated threonine residues were identified in PknL. Among them, we showed that the activation loop phosphorylated residues Thr173 and Thr175 were essential for the autophosphorylation activity of PknL. Phosphorylation of the activation loop Thr173 residue is also required for optimal PknL-mediated phosphorylation of Rv2175c. Together, our results indicate that phosphorylation of the PknL activation loop Thr residues not only controls PknL kinase activity but is also required for recruitment and phosphorylation of its substrate. Rv2175c was found to be phosphorylated when overexpressed and purified from Mycobacterium smegmatis as 2-DE indicated the presence of different phosphorylated isoforms. Given the presence of the dcw gene cluster in the close vicinity of the pknL/Rv2175c locus, and its conservation in all mycobacterial species, we propose that PknL/Rv2175c may represent a functional pair in the regulation of mycobacterial cell division and cell envelope biosynthesis.

A canonical FtsZ protein in Verrucomicrobium spinosum, a member of the Bacterial phylum Verrucomicrobia that also includes tubulin-producing Prosthecobacter species

BackgroundThe origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative.ResultsVerrucomicrobium spinosum, a member of Phylum Verrucomicrobia of domain Bacteria, has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The deduced amino acid sequence has sequence signatures and predicted secondary structure characteristic for FtsZ rather than tubulin, but phylogenetic trees and sequence analysis indicate that it is divergent from all other known FtsZ sequences in members of domain Bacteria. The FtsZ gene of V. spinosum is located within a dcw gene cluster exhibiting gene order conservation known to contribute to the divisome in other Bacteria and comparable to these clusters in other Bacteria, suggesting a similar functional role.ConclusionVerrucomicrobium spinosum has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The results suggest the functional as well as structural homology of the V. spinosum FtsZ to the FtsZs of other Bacteria implying its involvement in cell septum formation during division. Thus, both bacteria-like FtsZ and eukaryote-like tubulin cytoskeletal homologs occur in different species of the phylum Verrucomicrobia of domain Bacteria, a result with potential major implications for understanding evolution of tubulin-like cytoskeletal proteins and the origin of eukaryote tubulins.

P879 Cloning of a mdfA-like gene, encoding an effiux pump of an Acinetobacter baumannii clinical isolate

To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi).Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration–response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance.All 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 μg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 μg/mL.The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

Global RNA half-life analysis in Escherichia coli reveals positional patterns of transcript degradation.

Subgenic-resolution oligonucleotide microarrays were used to study global RNA degradation in wild-type Escherichia coli MG1655. RNA chemical half-lives were measured for 1036 open reading frames (ORFs) and for 329 known and predicted operons. The half-life of total mRNA was 6.8 min under the conditions tested. We also observed significant relationships between gene functional assignments and transcript stability. Unexpectedly, transcription of a single operon (tdcABCDEFG) was relatively rifampicin-insensitive and showed significant increases 2.5 min after rifampicin addition. This supports a novel mechanism of transcription for the tdc operon, whose promoter lacks any recognizable sigma binding sites. Probe by probe analysis of all known and predicted operons showed that the 5' ends of operons degrade, on average, more quickly than the rest of the transcript, with stability increasing in a 3' direction, supporting and further generalizing the current model of a net 5' to 3' directionality of degradation. Hierarchical clustering analysis of operon degradation patterns revealed that this pattern predominates but is not exclusive. We found a weak but highly significant correlation between the degradation of adjacent operon regions, suggesting that stability is determined by a combination of local and operon-wide stability determinants. The 16 ORF dcw gene cluster, which has a complex promoter structure and a partially characterized degradation pattern, was studied at high resolution, allowing a detailed and integrated description of its abundance and degradation. We discuss the application of subgenic resolution DNA microarray analysis to study global mechanisms of RNA transcription and processing.

Refinements of the Differential Display Reverse Transcription–Polymerase Chain Reaction Technique: Use of Oligo(dT)-Based Anchored Primers with Escherichia coli Messenger RNA Identifies a Salt-Induced Promoter in the dcw Gene Cluster

Differential display reverse transcription PCR (DDRT–PCR)2 is a popular technique used to identify differences in messenger RNA species expressed under different conditions (1–4). The use of this technique in prokaryotic systems has been greatly hampered by the lack of a poly(A) tail at the 39 end of these messages, resulting in the use of random primers to define both ends of the PCR products, in turn leading to lower probability of identifying any differentially expressed gene.

Cell division genes ftsQAZ in Escherichia coli require distant cis-acting signals upstream of ddlB for full expression.

A transcriptional reporter fusion has been introduced into the chromosomal ftsZ locus in such a way that all transcription that normally reaches ftsZ can be monitored. The new Phi(ftsZ-lacZ ) fusion yields four times more beta-galactosidase activity than a ddlB-ftsQAZ-lacZ fusion on a lambda prophage vector. A strongly polar ddlB ::Omega insertion prevents contributions from signals upstream of the ftsQAZ promoters and decreases transcription of the chromosomal Phi(ftsZ-lacZ ) fusion by 66%, demonstrating that around two-thirds of total ftsZ transcription require cis-acting elements upstream of ddlB. We suggest that those elements are distant promoters, and thus that the cell division and cell wall synthesis genes in the dcw gene cluster are to a large extent co-transcribed. The ddlB ::Omega insertion is lethal unless additional copies of ftsQA are provided or a compensatory decrease in FtsZ synthesis is made. This shows that ddlB is a dispensable gene, and reinforces the critical role of the FtsA/FtsZ ratio in septation. Using the new reporter fusion, it is demonstrated that ftsZ expression is not autoregulated.