Recombinant Pseudomonas syringae pv. syringae Protein CrcB homolog (crcB)

What are the key components of the carbon catabolite repression system in Pseudomonas syringae?

The carbon catabolite repression (CCR) system in Pseudomonas syringae primarily involves the interaction between the RNA-binding protein Crc and small non-coding RNAs (ncRNAs) including CrcZ and CrcX (formerly designated psr1 and psr2). These ncRNAs contain multiple "CA" motifs that bind to and sequester the Crc protein, thereby modulating its availability to regulate carbon metabolism. The system is ultimately controlled by the CbrA/CbrB two-component sensor-regulator system and the alternative sigma factor RpoN, which regulate the expression of these ncRNAs in response to different carbon sources and environmental conditions .

How do CrcZ and CrcX function in P. syringae compared to other Pseudomonas species?

In P. syringae, both CrcZ and CrcX function similarly to sequester the Crc protein, displaying functional redundancy. This differs from P. aeruginosa, which contains only CrcZ, and P. putida, which contains both CrcZ and CrcY. The regulation patterns also differ between species: in P. aeruginosa, succinate is a preferred carbon source that leads to low CrcZ levels, while in P. putida, succinate induces higher levels of CrcZ and CrcY. In P. syringae, fructose induces very high levels of both CrcZ and CrcX, suggesting species-specific adaptations to different carbon sources .

What is the genomic organization of the Crc regulatory ncRNAs in P. syringae?

In P. syringae pv. tomato DC3000, CrcZ (PSPTO_5668) is located between PSPTO_0964 and PSPTO_0963, while CrcX (PSPTO_5669) is located between PSPTO_1621 and PSPTO_1622. A third putative member, CrcY (psr3), is located between PSPTO_2739 and PSPTO_2740 but is disrupted by an insertion element in DC3000, rendering it non-functional. The transcript sizes of these ncRNAs range from 342 to 368 nucleotides, with CrcZ and CrcX containing five conserved "CA" regions and CrcY containing four .

How does carbon source affect the expression of CrcZ and CrcX?

The expression levels of CrcZ and CrcX in P. syringae DC3000 vary significantly depending on the carbon source. Compared to baseline expression in minimal medium, both ncRNAs show increased expression in the presence of fructose, glucose, mannitol, and citrate. Particularly notable is the very high expression observed when fructose is used as the sole carbon source. This carbon source-dependent regulation suggests that these ncRNAs play a crucial role in adapting the bacterium's metabolism to different environmental conditions .

What is the experimental evidence for functional redundancy between CrcZ and CrcX?

Growth experiments with P. syringae mutants demonstrate that deletion of both CrcZ and CrcX is required to observe growth defects in media containing mannitol, fructose, arabinose, or myo-inositol as the sole carbon source. Single deletion mutants of either CrcZ or CrcX do not show significant growth differences compared to wild-type. This indicates that these two ncRNAs have overlapping functions and can compensate for each other's absence. Interestingly, the double mutant also shows reduced growth with citrate, suggesting that unlike in P. aeruginosa, citrate may be a non-preferred carbon source for P. syringae DC3000 .

How do regulatory networks control CrcZ and CrcX expression?

Expression of CrcZ and CrcX in P. syringae is controlled by multiple regulatory factors. Quantitative RT-PCR experiments show that both ncRNAs are partially regulated by the alternative sigma factor RpoN. More significantly, their expression is strongly dependent on the CbrA/CbrB two-component system, with inactivation of cbrB resulting in dramatically reduced expression of both CrcZ and CrcX. Inactivation of cbrA also reduces expression, though less dramatically. This regulatory pattern indicates a complex control system that integrates multiple environmental signals to fine-tune carbon metabolism .

What recombineering techniques are effective for genetic manipulation of P. syringae?

Efficient recombineering in P. syringae has been achieved using the RecTE proteins identified from P. syringae pv. syringae B728a. These proteins are homologs of the RecET proteins from the Rac bacteriophage of E. coli. Experimental evidence shows that the RecT homolog alone is sufficient to promote recombination of single-stranded DNA oligonucleotides, while efficient recombination of double-stranded DNA requires expression of both RecT and RecE homologs. This system provides a valuable tool for making targeted gene disruptions in the P. syringae chromosome with higher efficiency than traditional methods .

What are the limitations of phage-based recombination systems in Pseudomonas species?

While phage-based recombination systems have worked exceptionally well in some bacterial species, adapting these systems to different bacteria, including Pseudomonas, can be problematic. Evidence suggests that these recombination systems have narrow species specificity, functioning robustly in one species but ineffectively in others. This limitation may be due to specific requirements for interactions between the recombinase and host-encoded factors. Only marginal success has been reported when using characterized phage recombination systems in Pseudomonas species, highlighting the need for species-specific recombineering tools .

What are the molecular mechanisms underlying RecTE-mediated recombination?

The RecTE recombination system typically involves the coordinated action of two key components: a 5′-to-3′ exonuclease (RecE) and a single-stranded DNA-annealing and strand invasion protein (RecT). In this process, RecE degrades one strand of double-stranded DNA, exposing 3′ single-stranded DNA ends. RecT then binds to these exposed ends, forming a protein-DNA filament that protects the substrate DNA and promotes annealing with the homologous genomic sequence. This RecA-independent recombination mechanism enables direct genetic engineering of chromosomal and episomal replicons .

How does temperature affect DNA repair mechanisms in P. syringae?

Temperature significantly impacts DNA repair mechanisms in Pseudomonas syringae, particularly at low temperatures. Research shows that all three subunits of the RecBCD enzyme (RecB, RecC, and RecD) are essential for DNA repair and growth at 4°C. Unlike in E. coli, where RecD is not essential for DNA repair, P. syringae requires all three subunits for effective function. Mutant analysis shows that viability of cells with mutations in any of these components drops drastically at 4°C, with accumulation of linear chromosomal DNA and shorter DNA fragments in higher amounts compared to cells grown at 22°C .

What is the role of the RecBCD pathway in cold adaptation of Antarctic P. syringae strains?

The RecBCD pathway plays a critical role in protecting Antarctic P. syringae strains from cold-induced DNA damage. Mutational analysis and genetic complementation studies establish that individual null-mutations of recC, recB, and recD genes, or deletion of the entire recCBD operon, lead to growth inhibition at low temperature and sensitivity to DNA-damaging agents like UV and mitomycin C. The pathway's function is critically dependent on the ATP-dependent helicase activities of both RecB and RecD subunits, while the nuclease activity of RecB appears to be less critical in vivo .

How can researchers experimentally assess the functional importance of different RecBCD components?

Researchers can assess the functional importance of RecBCD components through strategic mutational analysis. This includes creating null-mutations of individual genes (recC, recB, recD) or deleting the entire recCBD operon, then testing growth and viability at different temperatures. Additionally, creating specific mutations in functional domains provides deeper insights - mutations in the ATPase active sites of RecB (RecB K29Q) or RecD (RecD K229Q), or in the nuclease center of RecB (RecB D1118A and RecB Δnuc), can reveal which biochemical activities are most critical. Complementation studies using plasmid-expressed components from other species (e.g., E. coli recBCD) can further elucidate functional conservation across species .

How are Crc family ncRNAs distributed across Pseudomonas species?

Genomic analysis reveals variable numbers of Crc family members across Pseudomonas genomes. These ncRNAs comprise three main subfamilies: CrcZ, CrcX, and CrcY. All sequenced P. syringae strains contain the CrcX subfamily, which appears to be unique to this species. P. aeruginosa and P. mendocina strains typically contain only one Crc ncRNA (CrcZ), while P. putida harbors both CrcZ and CrcY. Analysis using computational models based on the gamma-150 motif has identified at least one Crc candidate ncRNA in nearly all Pseudomonas species, with P. geniculate being a notable exception .

What experimental approaches can be used to identify and characterize novel ncRNAs in bacterial genomes?

To identify and characterize novel ncRNAs in bacterial genomes, researchers can employ a multi-faceted approach: (1) Computational prediction using tools like CMFinder to identify conserved RNA motifs, followed by CMsearch to detect similar structures across genomes; (2) Transcriptional mapping to determine precise genomic boundaries using techniques like RNA-Seq and 5'RACE; (3) Expression analysis through qRT-PCR to evaluate ncRNA levels under different growth conditions or in different genetic backgrounds; (4) Functional analysis through targeted gene deletion and complementation studies; and (5) Structural analysis through both computational prediction and experimental methods such as chemical probing or crystallography .