Jogadhenu S. S. Prakash

How do bacteria sense and respond to low temperature

Rigidification of the membrane appears to be the primary signal perceived by a bacterium when exposed to low temperature. The perception and transduction of the signal then occurs through a two-component signal transduction pathway consisting of a membrane-associated sensor and a cytoplasmic response regulator and as a consequence a set of cold-regulated genes are activated. In addition, changes in DNA topology due to change in temperature may also trigger cold-responsive mechanisms. Inducible proteins thus accumulated repair the damage caused by cold stress. For example, the fluidity of the rigidified membrane is restored by altering the levels of saturated and unsaturated fatty acids, by altering the fatty acid chain length, by changing the proportion of cis to trans fatty acids and by changing the proportion of anteiso to iso fatty acids. Bacteria could also achieve membrane fluidity changes by altering the protein content of the membrane and by altering the levels of the type of carotenoids synthesized. Changes in RNA secondary structure, changes in translation and alteration in protein conformation could also act as temperature sensors. This review highlights the various strategies by which bacteria senses low temperature signal and as to how it responds to the change.

Planococcus antarcticus and Planococcus psychrophilus spp. nov. isolated from cyanobacterial mat samples collected from ponds in Antarctica

Abstract. Thirteen orange-pigmented bacteria associated with cyanobacterial mat samples collected from four different lakes in McMurdo, Antarctica, were isolated. Twelve of the isolates, which were coccoid in shape, were very similar and possessed all the characteristics of the genus Planococcus and represented a new species, which was assigned the name Planococcus antarcticus sp. nov. (CMS 26orT). Apart from the phenotypic differences, P. antarcticus differed from all reported species of Planococcus by more than 2.5% at the 16S rRNA gene sequence level. In addition, at the DNA–DNA hybridization level, it exhibited very little similarity either with P. mcmeekinii (30%–35%), P. okeanokoites (26%–29%), or CMS 53orT (15%–25%), the three species with which it is closely related at the rRNA gene sequence level (2.5%–2.9%). P. antarcticus also showed only 2.5% difference in its 16S rRNA gene sequence compared with the P. alkanoclasticus sequence. But it was distinctly different from P. alkanoclasticus, which exists only as rods, is mesophilic and phosphatase positive, can hydrolyze starch, cannot utilize succinate, glutamate, or glucose, and cannot acidify glucose. Most important, P. antarcticus and P. alkanoclasticus varied distinctly in their fatty acid composition in that C15:0, C15:1, C16:0, iso-C16:1, and C17:0 were present only in P. antarcticus but absent in P. alkanoclasticus. CMS 53orT, the thirteenth isolate, was also identified as a new species of Planococcus and was assigned the name Planococcus psychrophilus sp. nov. This species was distinctly different from all the reported species, including the new species P. antarcticus, with respect to a number of phenotypic characteristics. At the 16S rRNA gene sequence level, it was closely related to P. okeanokoites (98.1%) and P. mcmeekinii (98%), but with respect to the DNA–DNA hybridization, the similarity was only 35%–36%. The type strain of P. antarcticus is CMS 26orT (MTCC 3854; DSM 14505), and that of P. psychrophilus is CMS 530rT (MTCC 3812; DSM 14507).

Arthrobacter roseus sp. nov., a psychrophilic bacterium isolated from an Antarctic cyanobacterial mat sample

Strain CMS 90rT, a red-pigmented bacterium, was isolated from a cyanobacterial mat sample from a pond located in McMurdo, Antarctica. Based on its chemotaxonomic and phylogenetic properties, strain CMS 90r(T) was identified as a member of group I of Arthrobacter. It shared 16S rDNA similarity of 98% with Arthrobacter oxydans ATCC 14358T and Arthrobacter polychromogenes ATCC 15216T, while DNA-DNA similarities determined for these three organisms were less than 70%. It also differed from all 17 reported Arthrobacter species with A3alpha-variant peptidoglycan in that it possessed a unique peptidoglycan (Lys-Gly-Ala3) and contained galactose, glucose, ribose and rhamnose as cell-wall sugars. These data and the presence of diagnostic phenotypic traits support the description of CMS 90r(T) as a novel species of Arthrobacter, for which the name Arthrobacter roseus sp. nov. is proposed. The type strain is strain CMS 90r(T) (= MTCC 3712T = DSM 14508T).

Elevated temperature treatment induced alteration in thylakoid membrane organization and energy distribution between the two photosystems in Pisum sativum.

Two-week-old pea (Pisum sativum var. Arkal) plants were subjected to elevated temperature (38 °C/42 °C) in dark for 14−15 h. The effect of heat treatment on light- induced phosphorylation of LHCII and LHCII migration in the thylakoid membranes were investigated. The heat treatment did cause a substantial (more than two fold) increase in the extent of LHCII phosphorylation as compared to the control. Upon separation of appressed and nonappressed thylakoid fractions by digitonin treatment, the heat-treated samples showed a decrease in LHCII-related polypeptides from the grana stack (appressed region) over the control. Further, a small increase in the intensity of these (LHCII-related) bands was detected in stromal thylakoid fraction (non-appressed membranes). This suggests an enhanced extent of migration of phosphorylated LHCII from appressed to non-appressed regions due to in vivo heat treatment of pea plants. We also isolated the LHCII from control and heat treated (42 °C) pea seedlings. Analysis of CD spectra revealed a 5D6 nm blue shift in the 638 nm negative peak in heat treated samples suggesting alteration in the organization of Chl b in the LHCII macro-aggregates. These results suggest that in vivo heat stress not only alters the extent of migration of LHCII to stromal region, but also affects the light harvesting mechanism by LHCII associated with the grana region.

Psychrophilic Pseudomonas syringae requires trans-monounsaturated fatty acid for growth at higher temperature

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.

A novel Δ9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn−2 position of glycerolipids

Acyl-lipid desaturases are enzymes that convert a C-C single bond into a C=C double bond in fatty acids that are esterified to membrane-bound glycerolipids. Four types of acyl-lipid desaturase, namely DesA, DesB, DesC, and DesD, acting at the Delta12, Delta15, Delta9, and Delta6 positions of fatty acids respectively, have been characterized in cyanobacteria. These enzymes are specific for fatty acids bound to the sn-1 position of glycerolipids. In the present study, we have cloned two putative genes for a Delta9 desaturase, designated desC1 and desC2, from Nostoc species. The desC1 gene is highly similar to the desC gene that encodes a Delta9 desaturase that acts on C18 fatty acids at the sn-1 position. Homologues of desC2 are found in genomes of cyanobacterial species in which Delta9-desaturated fatty acids are esterified to the sn-2 position. Heterologous expression of the desC2 gene in Synechocystis sp. PCC 6803, in which a saturated fatty acid is found at the sn-2 position, revealed that DesC2 could desaturate this fatty acid at the sn-2 position. These results suggest that the desC2 gene is a novel gene for a Delta9 acyl-lipid desaturase that acts on fatty acids esterified to the sn-2 position of glycerolipids.

Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.

An RNA helicase, CrhR, regulates the low-temperature-inducible expression of heat-shock genes groES, groEL1 and groEL2 in Synechocystis sp. PCC 6803

The crhR gene for RNA helicase, CrhR, was one of the most highly induced genes when the cyanobacterium Synechocystis sp. PCC 6803 was exposed to a downward shift in ambient temperature. Although CrhR may be involved in the acclimatization of cyanobacterial cells to low-temperature environments, its functional role during the acclimatization is not known. In the present study, we mutated the crhR gene by replacement with a spectinomycin-resistance gene cassette. The resultant DeltacrhR mutant exhibited a phenotype of slow growth at low temperatures. DNA microarray analysis of the genome-wide expression of genes, and Northern and Western blotting analyses indicated that mutation of the crhR gene repressed the low-temperature-inducible expression of heat-shock genes groEL1 and groEL2, at the transcript and protein levels. The kinetics of the groESL co-transcript and the groEL2 transcript after addition of rifampicin suggested that CrhR stabilized these transcripts at an early phase, namely 5-60 min, during acclimatization to low temperatures, and enhanced the transcription of these genes at a later time, namely 3-5 h. Our results suggest that CrhR regulates the low-temperature-inducible expression of these heat-shock proteins, which, in turn, may be essential for acclimatization of Synechocystis cells to low temperatures.

Proteomics reveals a role for the RNA helicase crhR in the modulation of multiple metabolic pathways during cold acclimation of Synechocystis sp. PCC6803.

One of the earliest and largest transcriptional responses that occur during exposure of Synechocystis sp. PCC6803 to cold is the induction of the crhR RNA helicase transcript. We show that crhR deletion results in failure to cold acclimate: there is reduced growth at 24 °C and marked impairment of growth at 20 °C. 2D-DIGE, using five biological replicates, was used to analyze the proteomic differences between the wild-type and ΔcrhR strains grown at (1) 34 °C and (2) following transfer from 34 to 24 °C (cold-acclimation). Sixteen significantly differentially expressed proteins were identified between the two strains grown at 34 °C. Forty-three distinct proteins were identified that responded to cold-acclimation of the wild-type and 34 proteins for the mutant, with only 26 proteins common to both. A large proportion of the proteomic responses (76.5%) could not be predicted from published transcriptomic data. Only modest similarity is observed between proteomic and transcriptomic responses (r = 0.54-0.70). We propose functions for three previously hypothetical proteins. We suggest molecular targets for CrhR action and identify downstream regulated events in metabolism.

A novel transcriptional regulator, Sll1130, negatively regulates heat-responsive genes in Synechocystis sp. PCC6803

A conserved hypothetical protein, Sll1130, is a novel transcription factor that regulates the expression of major heat-responsive genes in Synechocystis sp. PCC6803. Synechocystis exhibited an increased thermotolerance due to disruption of sll1130. Δsll1130 cells recovered much faster than wild-type cells after they were subjected to heat shock (50°C) for 30 min followed by recovery at 34°C for 48 h. In Δsll1130 cultures, 70% of the cells were viable compared with the wild-type culture in which only 30% of the cells were viable. DNA microarray analysis revealed that in Δsll1130, expression of the heat-responsive genes such as htpG, hspA, isiA, isiB and several hypothetical genes were up-regulated. Sll1130 binds to a conserved inverted-repeat (GGCGATCGCC) located in the upstream region of the above genes. In addition, both the transcript and protein levels of sll1130 were immediately down-regulated upon shift of wild-type cells from 34 to 42°C. Collectively the results of the present study suggest that Sll1130 is a heat-responsive transcriptional regulator that represses the expression of certain heat-inducible genes at optimum growth temperatures. Upon heat shock, a quick drop in the Sll1130 levels leads to de-repression of the heat-shock genes and subsequent thermal acclimation. On the basis of the findings of the present study, we present a model which describes the heat-shock response involving Sll1130.