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Pseudomonas syringae Lz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential for P. syringae growth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPs enzyme displays distinct biochemical behaviors. Unlike E. coli RecBCD enzyme, the RecD subunit is indispensable for RecBCDPs function. The RecD motor activity is essential for the Chi-like fragments production in P. syringae, highlighting a distinct role for P. syringae RecD subunit in DNA repair and recombination process. Here, we demonstrate that the RecBCDPs enzyme recognizes a unique octameric DNA sequence, 5'-GCTGGCGC-3' (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 3'-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity in P. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest., Competing Interests: The authors have declared that no competing interests exist.

The Antarctic psychrotrophic bacterium Pseudomonas syringae was more sensitive to polymyxin B at a lower (4 degrees C) temperature of growth than at a higher (22 degrees C) temperature. The amount of hydroxy fatty acids in the lipopolysaccharides (LPS) also increased at the lower temperature. These changes correlated with the increase in fluidity of the hydrophobic phase of lipopolysaccharide aggregates in vitro.

A promoter-fusion study with a Tn 5-based promoter probe vector had earlier found that the hutU gene which encodes the enzyme urocanase for the histidine utilization pathway is upregulated at a lower temperature (4 degrees C) in the Antarctic psychrotrophic bacterium Pseudomonas syringae. To examine the characteristics of the urocanase gene and its promoter elements from the psychrotroph, the complete hutU and its upstream region from P. syringae were cloned, sequenced, and analyzed in the present study. Northern blot and primer extension analyses suggested that the hutU gene is inducible upon a downshift of temperature (22 to 4 degrees C) and that there is more than one transcription initiation site. One of the initiation sites was specific to the cells grown at 4 degrees C, which was different from the common initiation sites observed at both 4 and 22 degrees C. Although no typical promoter consensus sequences were observed in the flanking region of the transcription initiation sites, there was a characteristic CAAAA sequence at the -10 position of the promoters. Additionally, the location of the transcription and translation initiation sites suggested that the hutU mRNA contains a long 5'-untranslated region, a characteristic feature of many cold-inducible genes of mesophilic bacteria. A comparison of deduced amino acid sequences of urocanase from various bacteria, including the mesophilic and psychrotrophic Pseudomonas spp., suggests that there is a high degree of similarity between the enzymes. The enzyme sequence contains a signature motif (GXGX(2)GX(10)G) of the Rossmann fold for dinucleotide (NAD(+)) binding and two conserved cysteine residues in and around the active site. The psychrotrophic enzyme, however, has an extended N-terminal end.

A DNA-dependent RNA polymerase was purified from the Antarctic psychrotrophic bacterium Pseudomonas syringae. The RNA polymerase showed a typical eubacterial subunit composition with beta, beta', alpha2 and sigma subunits. The subunits cross-reacted with antibodies raised against holoenzyme and the individual subunits of the RNA polymerase of Escherichia coli. However, the enzyme was considered unique, since unlike the RNA polymerase of mesophilic E. coli it exhibited significant and consistent transcriptional activity (10-15%) even at 0 degrees C. But, similar to the enzyme from the mesophilic bacterium, the RNA polymerase from P. syringae exhibited optimum activity at 37 degrees C. The study also demonstrates that the RNA polymerase of P. syringae could preferentially transcribe the cold-inducible gene cspA of E. coli only at lower temperatures (0-22 degrees C). The polymerase was also observed to be relatively more rifampicin-resistant during transcription at lower temperature.

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.

Transcriptional activity was monitored in cells of the Antarctic psychrotrophic bacterium Pseudomonas syringae (Lz4W), which does not grow above 30 degrees C. It was observed that the bacterium was capable of synthesising RNA at a temperature range of 0-37 degrees C, both in vitro and in vivo. The net incorporation of the radioactive precursor, [3H]uridine, into RNA was found to be affected at 37 degrees C. A pulse-chase experiment following a 32P labeling of RNA in vivo indicated that the ribosomal RNAs (rRNAs) degrade faster at and above 30 degrees C. It was also found that the increased ribonuclease (RNase) activity at high temperature might be responsible for this degradation. The attack on ribosomal RNAs by RNase took place after their assembly into ribosomal particles. It is suggested that the degradation of rRNAs at supraoptimal temperatures might be a detrimental factor for growth above 30 degrees C.

The antarctic psychrotrophic bacterium Pseudomonas syringae was mutagenised using a transposon Tn5-OT182 which facilitates identification of promoter fusions expressing the reporter gene (lacZ) for beta-galactosidase. Most mutants expressed beta-galactosidase both at optimal growth temperature (20-22 degrees C) and at low temperature (4 degrees C). But a small percentage of the mutants (approximately 5%) were unique in that they expressed beta-galactosidase activity predominantly at low temperature. One such mutant was found to have an insertion in the gene for urocanase (hutU) of the histidine utilisation (hut) operon. Direct assay of urocanase and histidase activity in wild-type cells of various antarctic psychrotrophic strains including P. syringae, P. fluorescens and P. putida also suggested that the hut operon is expressed at an elevated level at low temperature.

Temperature-dependent phosphorylation and dephosphorylation of membrane proteins was studied in vitro in a number of psychrotrophic Antarctic bacteria which grow between 0 and 30 degrees C. One of them, a Pseudomonas syringae isolate, was studied in detail and was found to have three membrane proteins of molecular mass 30, 65 and 85 kDa which were phosphorylated differently in response to low and high temperatures. The 65 kDa protein was phosphorylated only at lower temperatures (between 0 and 15 degrees C). The 30 kDa protein was phosphorylated more at higher temperatures and was possibly a histidine kinase. This protein was present in all the psychrotrophic Pseudomonas species studied and in Sphingobacterium antarcticus. A possible role for these proteins in sensing environmental temperature is proposed.

Phosphorylation of lipopolysaccharide (LPS) from a psychrotrophic bacterium, Pseudomonas syringae, from Antarctica was studied by using sucrose gradient-separated membrane fractions. The bacterium was found to possess an LPS kinase which could phosphorylate more LPS postsynthetically at higher temperatures. The phosphorylation was low at a lower temperature and was also found to occur in vivo. After phosphorylation of LPS in vitro, it was found that the major part of the radioactivity (> 85%) was associated with the core oligosaccharide region of the LPS. The phosphate groups of this region are probably involved in the binding of metal ions, which could be removed by EDTA. The cells grown at the lower temperature probably contained fewer divalent cations because of the smaller amount of phosphate and thereby were more sensitive to EDTA. The cells were also more sensitive to cationic antibiotics at the lower temperature. A possible role of this differential phosphorylation of LPS in modulating the function of the outer membrane as a permeability barrier in the psychrotroph is discussed.