Proteomics of life at low temperatures: trigger factor is the primary chaperone in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

The proteomes expressed at 4 degrees C and 18 degrees C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two-dimensional differential in-gel electrophoresis, showing that translation, protein folding, membrane integrity and anti-oxidant activities are upregulated at 4 degrees C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl-prolyl cis-trans isomerase activity. This suggests that protein folding at low temperatures is a rate-limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat-shock proteins) are downregulated at 4 degrees C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33 degrees C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature-dependent and requires near-zero temperature to stably bind a model-unfolded polypeptide.

Life in the cold: a proteomic study of cold-repressed proteins in the antarctic bacterium pseudoalteromonas haloplanktis TAC125.

The proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis were compared using two-dimensional differential in-gel electrophoresis with special reference to proteins repressed by low temperatures. Remarkably, the major cold-repressed proteins, almost undetectable at 4°C, were heat shock proteins involved in folding assistance.

Improving Production of Protease from Pseudoalteromonas sp. CSN423 by Random Mutagenesis.

Pseudoalteromonas sp. CSN423, a marine strain, can express a major protease designated as E423 and it was secreted into the supernatant. To improve the protease E423 yield, Pseudoalteromonas sp. CSN423 was subjected to mutagenesis using UV irradiation. Mutant strain with 5.1-fold higher protease yield was isolated and named as Pseudoalteromonas sp. CSN423-M. Three protease bands were detected by zymography with casein as substrate, and results of mass spectrometry (MS) showed that two lower molecular weight protein bands were the same protease but with different mature forms. The entire protease operon was sequenced and no mutation was found. Mutant strain-associated changes of expression levels of protease synthesis and secretion-related genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Mutant strain had higher expression of e423 than wild-type strain. Such result was consistent with protease activity profiles. Moreover, the mutant strain had higher transcriptional levels of citrate synthase (cs), α-ketoglutarate decarboxylase (kgd), cytochrome c oxidase subunit I (coxI), tolC, hlyD (membrane protein), luxR3, luxO, and luxT (transcriptional regulator). However, hexokinase (hk), pyruvate dehydrogenase E1 (pd-e1), epsD (membrane protein), and luxR1 remained unchanged, and luxR2 decreased sharply in the mutant. These results suggested that the redox pathway was promoted in the mutant strain, and LuxR family transcriptional regulators in Pseudoalteromonas spp. may play some role in regulating protease expression. Meanwhile, the secretion of extracellular protease was closely related to ABC transport system. These results may shed some light on the molecular mechanism underlying higher yield of protease E423 from Pseudoalteromonas sp. CSN423-M.

Molecular level damages of low power pulsed laser radiation in a marine bacterium Pseudoalteromonas carrageenovora.

AIM: To study the molecular level damages in a marine bacterium, Pseudoalteromonas carrageenovora, exposed to low power pulsed laser radiation from an Nd:YAG laser. METHODS AND RESULTS: The laser damages in bacterial DNA were monitored by studying the formation of apurinic/apyrimidinic (AP) sites. Molecular probe kits were used for this purpose. Occurrence of lesions in the cell walls was monitored under a transmission electron microscope (TEM). The results showed that laser radiation significantly increased the number of AP sites in the bacterial DNA. This increase corresponded to the laser fluence (J cm(-2)) and to the duration of laser irradiation. TEM observation showed the occurrence of lesions in bacterial cell walls upon laser irradiation. CONCLUSIONS: It is concluded that bacteria exposed to laser irradiation suffers DNA damages and resulted in broken cell walls. These events led to bacterial mortality. These are in addition to the mechanisms reported earlier such as the photochemical reactions occurring inside the cells upon exposure to low power laser. SIGNIFICANCE AND IMPACT OF THE STUDY: These results help us to understand the mechanisms of bacterial mortality on exposure to low power pulsed laser irradiation and are useful in formulating a laser treatment strategy to kill bacteria.

In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength.

We studied the laser ablation of laboratory-developed biofilm on titanium and glass surfaces. Specifically, Pseudoalteromonas carrageenovora, a marine biofilm forming bacterium was used to generate laboratory biofilm. Two fluences, 0.05 and 0.1 J/cm(2) and three durations of irradiation, 30 s, 5 min, and 10 min were tested using an Nd;YAG laser of 532 nm wavelength (in the green light area). Nonirradiated coupons with biofilm served as control. The biofilm removal efficiency increased with the increase in laser fluence and duration of irradiation. The maximum biofilm area cover on control coupons of glass and titanium was 62.5 and 76.0%, respectively. Upon irradiation with fluence 0.1 J/cm(2) for the very short duration of 30 s, this reduced to 5.6 and 12.4% and at 10 min to 2.17 and 0.7% on glass and titanium coupons, respectively, while the controls did not show any reductions (62.5 and 76.0% respectively, for glass and titanium coupons). The biofilm TRC (Total Resuscitated Cells) reduction during this period was even more prominent than the area cover, indicating that the remaining biofilm portions on coupons after irradiation were largely composed of dead bacterial cells. The TRC in the irradiation chamber medium for short durations of irradiation showed a significant increase, indicating that the laser irradiation removed live bacteria from the biofilm. The re-growth of the resuscitated cells showed they could grow like the control cells but with a significant lag. The laser's efficiency in the removal of biofilm was better seen on titanium coupons than on glass. Our results showed that a low-power pulsed laser irradiation could be used to remove biofilm formed on hard surfaces.

In vitro laser ablation of natural marine biofilms.

We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons. Natural biofilms with thicknesses of 79.4 +/- 27.8 microm (titanium) and 107.4 +/- 28.5 microm (glass) were completely disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm2). Laser irradiation significantly reduced the number of diatoms and bacteria in the biofilm (paired t test; P < 0.05). The removal was better on titanium than on glass coupons.

Impact of pulsed Nd:YAG laser on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora: significance of physiological status.

The impact of pulsed Nd:YAG (neodymium-doped yttrium/aluminium garnet) laser irradiation on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora during two growth stages (log phase and stationary phase) and under two stresses (reduced temperature and nutrient limitation) was investigated. Bacteria were exposed to a laser fluence of 0.1 J x cm(-2) for 5, 10, and 15 min with a peak power of 20 MW x cm(-2), a pulse width of 5 ns, and an average power of 1 W x cm(-2) with a repetition rate of 10 Hz. The mortality of bacteria immediately after the irradiation as well as after a set period of time was determined. Mortality was higher among log-phase bacteria (72%) than bacteria in the stationary phase (51%) and those grown under nutrient limitation (51%). Bacteria grown at reduced temperature had a mortality of 49%. However, the differences in cell density of log-phase, stationary-phase, nutrient-limited, and low-temperature irradiated samples compared with controls after 5 h of incubation were 96, 93, 94, and 86%, respectively. The mortality values suggest that the same laser fluence has different degrees of effectiveness, depending on the physiological state of the bacteria.

Inhibition of bacterial attachment by pulsed Nd:YAG laser irradiations: an in vitro study using marine biofilm-forming bacterium Pseudoalteromonas carrageenovora.

The effect of low mean power laser irradiations with short pulse duration from an Nd:YAG (neodymium-doped yttrium aluminium garnet) laser on a marine biofilm-forming bacterium, Pseudoalteromonas carrageenovora, was investigated in the laboratory. Laser-irradiated bacteria were tested for their ability to attach on nontoxic titanium nitride (TiN) coupons with nonirradiated bacteria as the reference. Two durations of irradiation were tested, 10 and 15 min. Bacterial attachment was monitored after 20 min, 40 min, and 1 h of irradiation. The average laser fluence used for this study was 0.1 J/cm(2). The area of attachment of the irradiated bacteria was significantly less than the reference for both durations of irradiation. The growth of irradiated bacteria showed a longer lag phase than the nonirradiated sample, mainly due to mortality in the former. The bacterial mortality observed was 23.4 +/- 0.71 and 48.6 +/- 6.5% for 10- and 15-min irradiations, respectively. Thus, the results show that low-power pulsed laser irradiations resulted in a significant bacterial mortality and a reduced bacterial attachment on nontoxic hard surfaces.

Laser impact assessment in a biofilm-forming bacterium Pseudoalteromonas carrageenovora using a flow cytometric system.

Impact by pulsed laser irradiations from an Nd:YAG laser on the marine biofilm-forming bacterium Pseudoalteromonas carrageenovora has been studied using a flow cytometric system. The biofilm-forming bacteria in the planktonic state have been irradiated while flowing, and the mortality and bacterial attachment have been determined by exposing TiN coupons in the system. Coupons suspended in the non-irradiated bacterial flow were treated as the control. The fluence used in the study was 0.1 J/cm(2). Three flow rates (14, 28, and 42 cm/min) and two exposure durations (15 and 30 min) were tested. The results showed the increase in bacterial mortality with the decrease in flow rate. The maximum mortality of 27.5% was observed when the flow rate was 14 cm/min. The bacterial attachment increased with the increase in flow rate and exposure duration. The area of bacterial attachment on the experimental coupons exposed to the irradiated sample was significantly lesser than that for the nonirradiated sample. The results thus show in a flowing system, low power pulsed laser irradiations could reduce the bacterial attachment even though it did not cause significant mortality.