In nature, microorganisms have to adapt to long-term stressful conditions often with growth limitations. However, little is known about the evolution of the adaptability of new bacteria to such ...environments. Pseudomonas aeruginosa, an opportunistic pathogen, after natural evaporation of seawater, was shown to be trapped in laboratory-grown halite crystals and to remain viable after entrapment for years. However, how this bacterium persists and survives in such hypersaline conditions is not understood.
In this study, we aimed to understand the basis of survival, and to characterise the physiological changes required to develop salt tolerance using P. aeruginosa as a model. Several clones of P. aeruginosa were rescued after 14 years in naturally evaporated marine salt crystals. Incubation of samples in nutrient-rich broth allowed re-growth and subsequent plating yielded observable colonies. Whole genome sequencing of the P. aeruginosa isolates confirmed the recovery of the original strain. The re-grown strains, however, showed a new phenotype consisting of an enhanced growth in growing salt concentration compared to the ancestor strain. The intracellular accumulation of K
was elicited by high concentration of Na
in the external medium to maintain the homeostasis. Whole transcriptomic analysis by microarray indicated that 78 genes had differential expression between the parental strain and its derivative clones. Sixty-one transcripts were up-regulated, while 17 were down-regulated. Based on a collection of single-gene knockout mutants and gene ontology analysis, we suggest that the adaptive response in P. aeruginosa to hyper-salinity relies on multiple gene product interactions.
The individual gene contributions build up the observed phenotype, but do not ease the identification of salinity-related metabolic pathways. The long-term inclusion of P. aeruginosa in salt crystals primes the bacteria, mediating a readjustment of the bacterial physiology to growth in higher salt concentrations. Our findings provide a starting point to understand how P. aeruginosa, a relevant environmental and pathogenic bacterium, survives to long-term salt stress.
The pathogenic bacterium Salmonella enterica serovar Typhimurium elicits a variety of genetic programs to adapt to stress conditions encountered within hostile environments such as host phagocytes ...and preserved food.
In this work, differential display (DD) methodology was used to investigate the effect of one month starvation in a salty microcosm (0.5 M NaCl) on transcript profiling in a Salmonella Typhimurium LT2 strain. cDNA fragments resulting from differentially expressed mRNA were eluted from the gel, re-amplified, cloned, and then sequenced.
A total of 21 differentially expressed bands were detected by DD reverse transcription-polymerase chain reaction (RT-PCR). However, only 12 of them were successfully identified as upregulated genes in stressed cells. Based on the sequencing data and BLAST analysis, these genes were sopA, ssaD, yhhK, gmK, cspC, uspA, ompR, phoP, stcC, fimA, acrA,and yehZ. As a confirmation of the differential expression, RT-PCR was carried out using a set of specific primers. Remarkably, the expression levels of these genes were significantly increased in starved bacteria compared to standard laboratory conditions.
Our results indicate that the starvation of Salmonella Typhimurium over one month in a salty microcosm changes the expression of stress proteins, response regulator in a two-component system, outer membrane proteins, effector proteins translocated by Salmonella pathogenicity island SPI1 and SPI2 type III secretion systems (TTSS), several metabolic enzymes, efflux pumps, and transport proteins. This suggests that the expression of the identified genes is important for the response of this pathogen to starvation in salt.
In this study, three lactic acid bacteria (LAB), isolated from barley, traditional dried meat and fermented olive were characterized and tested for their anti-bacterial and anti-biofilm activities ...against oral bacteria.
Our results revealed that the tested LAB were γ-hemolytic and were susceptible to four antibiotics. All the strains were resistant to low pH, bile salt, pepsin and pancreatin. Furthermore, FB2 displayed a high aut-oaggregative phenotype (99.54%) while FF2 exhibited the best co-aggregation rate. Concerning the microbial adhesion to solvent, FB2 was the most hydrophobic strain (data obtained with chloroform and n-hexadecane). In addition Pediococcus pentosaceus FB2 and Lactobacillus brevis FF2 displayed a significant inhibitory effect against Streptococcus salivarius B468 (MIC = 10%). Moreover the selected strains were able to inhibit biofilm formation of Bacillus cereus ATCC14579 (MBIC50 = 28.16%) and S. salivarius B468 (MBIC50 = 42.28%).
The selected LAB could be considered as candidate probiotics for further application in functional food and mainly in the prevention of oral diseases.
•Three lactic acid bacteria (LAB), isolated from barley, traditional dried meat and fermented olive were characterized.•The selected LAB were γ-hemolytic, resist to low pH, bile salt, pepsin and pancreatin.•A significant inhibitory effect of P. pentosaceus FB2 and L. brevis FF2 against S. salivarius B468 was notice.•The selected strains were able to inhibit biofilm formation of B. cereus and S. salivarius B468.
Even with advances in molecular cloning and DNA transformation, new or alternative methods that permit DNA penetration in Salmonella enterica subspecies enterica serovar Typhimurium are required in ...order to use this pathogen in biotechnological or medical applications. In this work, an adapted protocol of bacterial transformation with plasmid DNA based on the “Yoshida effect” was applied and optimized on Salmonella enterica serovar Typhimurium LT2 reference strain. The plasmid transference based on the use of sepiolite as acicular materials to promote cell piercing via friction forces produced by spreading on the surface of a hydrogel. The transforming mixture containing sepiolite nanofibers, bacterial cells to be transformed and plasmid DNA were plated directly on selective medium containing 2% agar. In order to improve the procedure, three variables were tested and the transformation of Salmonella cells was accomplished using plasmids pUC19 and pBR322. Using the optimized protocol on Salmonella LT2 strain, the efficiency was about 105 transformed cells per 109 subjected to transformation with 0.2μg plasmid DNA. In summary, the procedure is fast, offers opportune efficiency and promises to become one of the widely used transformation methods in laboratories.
•A protocol of Salmonella transformation using sepiolite nanofibers was optimized.•The tested variables are: amount of sepiolite, number of cells and spreading time.•Using the general protocol, the efficiency was about 5×105 per 1μg of plasmid.•The method is simple, rapid, and inexpensive and does not require competent cell preparation.
Listeria monocytogenes
is a food-borne pathogen of humans and other animals. The striking ability to survive several stresses usually used for food preservation makes
L. monocytogenes
one of the ...biggest concerns to the food industry. This ubiquity can be partly explained by the ability of the organism to grow and persist at very low temperatures, a consequence of its ability to accumulate cryoprotective compound called osmolytes. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the stress response genes
opuCA
and
betL
(encoding carnitine and betaine transporters, respectively) and the housekeeping gene
16S rRNA
. Assays were conducted on mid-exponential phase
L. monocytogenes
cells exposed to conditions reflecting cold and freezing stress, conditions usually used to preserve foods. We showed that expression of the two cold-adapted genes encoded the transporters of the cryoprotectants carnitine and betaine in ATCC 19115 and the food-isolated
L. monocytogenes
S1 is induced after cold and freezing stress exposure. Furthermore, transcriptional analysis of the genes encoding
opuCA
and
betL
revealed that each transporter is induced to different degrees upon cold shock of
L. monocytogenes
ATCC 19115 and S1. Our results confirm an increase in carnitine uptake at low temperatures more than in betaine after cold-shocked temperature compared to the non-stress control treatment. It was concluded the use of carnitine and betaine as cryoprotectants is essential for rapid induction of the tested stress response under conditions typically encountered during food preservation.
Three bacterial strains (TE1, TD3 and FB2) were isolated from date palm (degla), pistachio and barley. The presence of nitrate reductase (narG) and nitrite reductase (nirS and nirK) genes in the ...selected strains was detected by PCR technique. Molecular identification based on 16S rDNA sequencing method was applied to identify positive strains. In addition, the D-optimal mixture experimental design was used to optimize the optimal formulation of probiotic bacteria for denitrification process.
Strains harboring denitrification genes were identified as: TE1, Agrococcus sp LN828197; TD3, Cronobacter sakazakii LN828198 and FB2, Pedicoccus pentosaceus LN828199. PCR results revealed that all strains carried the nirS gene. However only C. sakazakii LN828198 and Agrococcus sp LN828197 harbored the nirK and the narG genes respectively. Moreover, the studied bacteria were able to form biofilm on abiotic surfaces with different degree.
Process optimization showed that the most significant reduction of nitrate was 100% with 14.98% of COD consumption and 5.57 mg/l nitrite accumulation. Meanwhile, the response values were optimized and showed that the most optimal combination was 78.79% of C. sakazakii LN828198 (curve value), 21.21% of P. pentosaceus LN828199 (curve value) and absence (0%) of Agrococcus sp LN828197 (curve value).
•Three probiotic strains were identified as Agrococcus sp. LN828197, C. sakazakii LN828198 and P. pentosaceus LN828199.•These strains harbored denitrifing genes.•The most predictable reduction of nitrate was 100% with 14.98% of COD consumption and 5.57 mg/l nitrite accumulation.•The D-optimal mixture experimental design is the an tools for optimizing the proportion combination in each assay.
The adaptive response of bacteria to stress is variable owing to the effects of various environmental factors and the time of exposure to stress conditions. We have studied the morphological and ...biochemical characters of resuscitated Pseudomonas aeruginosa ATCC 27853 that had been incubated for 14 years in natural sterilized seawater. The resuscitated cells were identified by PCR analysis of the internal transcribed spacer region. The biochemical profile of the resuscitated cells was partially inactive according to the API-20NE system. The cells had lost the ability to reduce nitrate and hydrolyse gelatin but acquired urease activity. In addition, the atomic force micrographs showed a reduction in cell size and an evolution to coccoid shapes. After incubation in a rich medium, the resuscitated cells regained their initial biochemical characters and their original rod shape. The differential display “DDRT-PCR” technique was used to determine the effects of stress on gene expression in this P. aeruginosa strain. The genes algU and iscR were successfully identified in stressed P. aeruginosa ATCC 27853 as two differentially expressed gene fragments. Our results demonstrate that the DDRT-PCR technique can be a useful tool in gene expression analysis studies looking at the stress response in P. aeruginosa.
The development of antibiotic resistance in the opportunistic pathogen
Pseudomonas aeruginosa
is a major cause of the pathogen’s morbidity and is strongly correlated with the biofilm formation. ...Motility and adherence capacity in long-term stressed cells have not been extensively analyzed even though
P. aeruginosa
considered a model organism for the study of biofilm formation. In this investigation,
P. aeruginosa
ATCC 27853 strain has been stored for 12 months in LB broth with 0.5 M NaCl. Several experiments demonstrated that the strain recovery from the salty microcosm had the ability to increase the biofilm formation and to reduce motility comparing with that of the original strain. To identify genes involved in the regulation of biofilm and/or in stress response by the recovered
P. aeruginosa
, differential display “DDRT-PCR” technique was used. The genes
speD
and
ccoN2
, coding, respectively, for an S-adenosylmethionine decarboxylase and Cbb3-type cytochrome oxidase, were identified in recovered strain of
P. aeruginosa
ATCC 27853 as two differentially expressed gene fragments. A comparison of the biofilm produced by the wild-type strain PA14 and the transposon insertion mutant for
speD
gene suggested that spermidine has a potential role in the adaptive response in
P. aeruginosa
incubated in long-term stress conditions.
Current protocols of recombinant DNA research, including gene cloning and complementation, quantification of gene expression and tagging with reporter proteins, are usually limited by the ...availability of effective bacteria transformation tools different from Escherichia coli. This is particularly relevant with respect to the Pseudomonas species due to their biotechnological and sanitary importance. Here, we describe an optimized and efficient plasmid transference protocol based on the Yoshida effect, a method that relies on DNA uptake mediated by friction forces. The main advantages of this method are: (i) no competent cell preparation is needed, (ii) cells in any physiological state can be used, (iii) the procedure is performed directly on agar plates and (iv) the protocol, which is neither time-consuming nor labor-intensive, offers good efficiency. This approach promises to become the gold standard for day to day genetic manipulation in Pseudomonas.
► Efficient plasmid transformation protocol for Pseudomonas sp. ► The method do not require competent cell preparation. ► Cells at any physiological state can be used. ► The procedure is performed directly on agar plates. ► Advantages for high throughput screenings.
PDF
