Identification of genes involved in long-term survival in Pseudomonas aeruginosa PAO 1

Pseudomonas aeruginosa is a remarkably adaptable organism which is able to survive and persist under a broad range of environmental conditions. The traditional views of bacterial growth phases usually come from standard laboratory cultures which can be different from natural conditions. In many natural environments, bacteria could maintain a long-term surviving status. To understand bacterial long-term survival, a luxCDABE-based random promoter library of P. aeruginosa was used to screen genes persistently expressed in the long-term culture of which 45 of such genes were identified. Among them, 13 genes encode different enzymes and are involved in different metabolic processes. Eight genes encode transcriptional regulators or two-component systems. In addition, a large portion of the identified genes encode hypothetical proteins with unknown function. Six of these unknown genes (PA1216, PA2827, PA0550, PA0256, PA0057 and PA4578) were selected and gene knockout mutants were constructed. All of these mutants exhibited reduced competitiveness than the wild-type PAO1 in the long-term competition assay, suggesting that these genes may play important roles for the long-term survival in P. aeruginosa.


INTRODUCTION
Traditionally, bacteria are mostly studied in batch growth conditions, where four growth phases, the lag phase, exponential phase, stationary phase and death phase are recognized.Bacteria experience a feast and famine cycle in these conditions.While being an essential methodology with which a great deal have been leant in bacterial physiology and genetics, the physiology and genetics in these conditions unavoidably do not always reflect those of bacteria in naturally environments.
In previous studies, the fifth phase of the bacterial life cycle, long-term stationary phase, has been identified in the long-term laboratory culture (Finkel, 2006).During the transition from exponential to stationary phase, cells undergo a variety of morphological and physiological changes that assist them in survival as preferred nutrient pools are depleted.Upon continued incubation, bacteria are able to survive for exceptionally long periods (years) without input of exogenous nutrients (Finkel et al., 2000).In many natural environments, bacteria probably exist in conditions more akin to these of long-term stationary phase culture.
Opportunistic pathogen Pseudomonas aeruginosa, that are capable of thriving in diverse environments ranging from water and soil to plants and animal tissues can cause acute or chronic infections in humans (Yahr and Parsek, 2006).Numerous studies of this pathogen have been routinely obtained using standard laboratory cultures.In many natural environments, the nutrients are probably limited and the bacteria do not go through a boom and burst cycle, and instead they maintain a long term surviving status where bacteria remain viable but not  (Hoang et al., 1998) pZ1918-lacZGm Source plasmid of Gm r cassette; Gm r (Schweizer, 1993) pRK2013 Broad-host-range helper vector; Tra + , Kn r (Ditta et al., 1980) pMS402 Expression reporter plasmid carrying the promoterless luxCDABE gene; Kn r , Tmp r (Duan et al., 2003) pEX-1216 PA1216 knockout plasmid, pEX18Ap with upstream region, Gm In order to understand the long-term survival, a lux-based random promoter library of P. aeruginosa constructed by Duan et al. (2003) was used to screen persistently expressed genes in the long-term culture according to luminescence intensity.Forty five (45) genes were identified from more than 3000 clones of the promoter library.A large portion of the identified genes encode hypothetical proteins with unknown function.Long-term competition assays showed that six of such unknown genes (PA1216, PA2827, PA0550, PA0256, PA0057 and PA4578) tested may play important roles in long-term survival in P. aeruginosa.

Bacterial strains, plasmids and culture conditions
Bacterial strains and plasmids used in this study are described in Table 1.P. aeruginosa and Escherichia coli were routinely grown on

Promoter screening and clustering
A lux-based random promoter library of P. aeruginosa ATCC27853 constructed by Duan et al. (2003) was used to screen persistent expression genes.The luminescence of each promoter clone was measured as counts per second (cps) of light production in a Wallac Victor 2 model 1450 multilabel counter (Perkin-Elmer Life Sciences) during long-term culture in multiwell plates.The promoter clustering was performed according to the similarity in their expression profiles using the CLUSTER program and visualized using TREEVIEW.

Sequence analysis of the persistent expression promoters
A subset of persistently expressed promoters was polymerase chain reaction (PCR) amplified using primers pZE.05 and pZE.06 (Table 2), which flank the BamHI site of pMS402.The PCR products were sequenced, and DNA sequences obtained were compared with the P. aeruginosa chromosome sequences (http://www.pseudomonas.com/)to verify related genes.

Construction of gene knockout mutants
For construction of gene knockout mutants, the previously described sacB-based strategy was used (Hoang et al., 1998).The DNA regions of the target genes were PCR amplified using primers listed in Table 2. Restriction sites were incorporated into the primers to facilitate cloning.The PCR products obtained were digested with restriction enzymes; and then cloned into pEX18Ap.The DNA fragment containing the Gmr-lacZ from pZ1918-lacZGm (Schweizer, 1993) was inserted into the target genes.Gene knockout mutants were obtained using the triparental mating procedure in which the strain carrying the helper plasmid pRK2013 (Ditta et al., 1980) was used together with the donor and recipient.The resultant mutants were verified by PCR.

Competition assay
A competition assay was used to compare the long-term survivability of the wild type and the mutants.Overnight cultures of P. aeruginosa strains were adjusted to an OD600 of 0.5, then 1 ml adjusted cultures of different mutant strains were co-inoculated into 50 ml fresh LB medium with the wild-type PAO1 at a ratio of 1:1 respectively.These co-cultures were incubated at 37°C with shaking (200 rpm) for 20 days.Total cell counts and mutant numeration were performed on LB agar or antibiotic selective agar after 1 day, 3, 14, and 20 days incubation.

Screening of P. aeruginosa genes persistently expressed in long-term culture
In order to investigate the bacterial state in long-term survival, a lux-based random promoter library of P. aeruginosa was used to screen persistently expressed genes in the long-term culture.The random promoter library was constructed by a low-copy-number plasmid pMS402 carrying a promoterless luxCDABE reporter gene cluster.The activity of individual promoter was represented by the amount of light generated by the clone containing the construct.By measuring luminescence in a multilabel plate counter, the P. aeruginosa library can be screened under different conditions to identify differentially regulated genes.
Using this method, we screened more than 3000 P. aeruginosa clones for persistently expressed promoters.During the long-term culture in multiwell plates, most of the genes were only expressed in the first few days.A number of genes were found to express further for more than 20 days.Actually, the other expression peak appeared in the seventeenth day (Figure 1, D09).Certain genes even had enhanced expression after 20 days incubation (Figure 1, D06).We hypothesized that these persistently expressed genes may have important function for bacterial long-term survival.The clones which had persistent expression profiles were picked out for further study.

Characterization of the persistent expression genes in P. aeruginosa
The persistent expression promoters were sequenced and compared with the annotated P. aeruginosa PAO1 genome to identify relevant genes.Table 3 lists the 45 operons that are persistently expressed during the long-term incubation.These genes can be classified into five groups by function.
Among the genes that were persistently expressed in long-term culture, many genes were involved in metabolic process (Table 3, the first group).Two genes, argF and metX, were involved in amino acid biosynthesis and metabolism (Itoh et al., 1988;Bourhy et al., 1997).codA, which encodes cytosine deaminase (Danielsen et al., 1992), participates in nucleotide metabolic process.Ten other genes which encode different enzymes were involved in different metabolic processes.
Eight of the identified genes encode transcriptional regulators or two-component systems (Table 3, the second group).mucC encodes a positive regulator for alginate biosynthesis (Boucher et al., 1997).parR and cbrA are two-component system genes which are involved in regulating antibiotic resistance and utilization  of carbon and nitrogen sources, respectively (Nishijyo et al., 2001;Fernandez et al., 2010).All these regulatory genes may participate in regulating the expression of genes required for long-term survival in P. aeruginosa.Two genes in the third group encode probable transporter proteins.Other known genes are collected as the fourth group, including pilC which encodes the still frameshift type 4 fimbrial biogenesis protein PilC (Nunn et al., 1990), pchD encodes the pyochelin biosynthesis protein PchD (Serino et al., 1997), and pasP encodes a protease PasP (Marquart et al., 2005).
In addition to the above genes, 18 of the identified genes encode hypothetical proteins with unknown function.This number represents 40% of the 45 characterized promoters.These data imply that some of the unknown genes may only be functional under long-term survival conditions.

Loss of viability of mutants in long-term co-culture with wild-type strain
To investigate the function of these persistently expressed genes in bacterial long-term survival, six of these unknown genes (PA1216, PA2827, PA0550, PA0256, PA0057 and PA4578) were selected for further investigation.Gene knockout mutants were generated by inserting a Gm r -lacZ cassette into these genes.A long-term competition assay was used to test the competitiveness of these mutants.Different mutant strains were co-inoculated with the wild-type PAO1 at a ratio of 1:1.These co-cultures were incubated at 37°C for 20 days.The survival rates of parental and mutant strains were calculated after one, three, 14, and 20 days incubation.
In the first three days, there were no significant difference in survival rates between the wild-type and the mutants.However, after 14 days incubation, distinct difference in survival rates was observed for the PA2827 mutant and PAO1 (Figure 2A).PA0550 mutant also exhibited obviously reduced competitiveness than the wild-type strain (Figure 2B).Moreover, the rest of the mutants displayed decreased competitiveness in different degrees (data not shown).The results suggest that these persistently expressed genes may indeed play a role for the long-term survival in P. aeruginosa.

DISCUSSION
P. aeruginosa strains are found in various environmental habitats as well as in animal and human hosts.The high potential for adaptation to new environmental conditions is mostly due to the large genomes of P. aeruginosa strains (Stover et al., 2000).The physiology and genetics of P. aeruginosa strains are usually leant through conventional laboratory culture.Some of the genes which do not express in normal conditions could always be ignored.
In an attempt to investigate this problem, we set up a long-term laboratory culture condition without continuously input of exogenous nutrients.A luxCDABE-based random promoter library of P. aeruginosa was used to screen persistent expression genes in the long-term culture.Using this method, 45 genes which can continuously express during long-term incubation were identified from the promoter library.Lots of genes encoding metabolic enzymes, transcriptional regulators and two component regulatory systems were found in this process.Successful adaptation of living organisms to different niches depends on their ability to regulate gene expression as specified by the needs of the environment (Stock et al., 2000).Persistently expressing genes involved in metabolism and transcriptional regulation may enable the bacteria to adapt to this infertile environment.In addition to these well-characterized genes, a large part of the identified genes encode hypothetical proteins.These unknown genes may have essential functions for bacterial long-term survival.
In order to investigate the functions of these persistent expression genes in long term survival, six of the unknown genes were selected for gene knockout mutagenesis.A long-term competition assay was used to test the competitiveness of these mutants.The survival rates of parental and mutant strains were compared during the long-term co-culture.As expected, all of these six mutants showed significant decrease in competitiveness.This result indicates that these persistent expression genes may play important roles in long-term survival in P. aeruginosa.

Figure 1 .
Figure 1.The expression profiles of clones D06 and D09 during the long-term culture.The Y-axis indicates cps value; the X-axis represents time course (h, hour; d, day).This experiment was repeated at least three times, and curves shown are representatives of similar results.

Figure 2 .
Figure 2. The survival rates of mutants and wild-type PAO1 during long-term culture.The Y-axis indicates survival rates; the X-axis represents time course.Error bars indicate one standard deviation.

Table 1 .
Strains and plasmids used in this study.
+ gene replacement vector with multiple-cloning site from pUC18; Ap r

Table 2 .
Primers used in this study.

Table 3 .
List of genes that persistently expressed in long-term culture.