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Legionella pneumophila is an aquatic bacterium that is also the agent of Legionnaires' disease pneumonia. Since L. pneumophila is transmitted directly from the environment to the lung, it is important to understand how legionellae survive at low temperatures. To identify genes that are needed for L. pneumophila growth at low temperature, we screened a population of mutagenized legionellae for strains that are specifically impaired for growth at 17°C. From the 7400 mutants tested, eleven displayed defects ranging from ca. 10-fold to a complete inability to grow at the low temperature. PCR and sequence analysis were then utilized to identify the genes whose loss had compromised growth. The proteins thereby implicated in low-temperature growth included components of the type II secretion system (LspE, LspG, LspH), a lipid A biosynthetic enzyme (LpxP), a ribonuclease (RNAse R), an RNA helicase (CsdA/DeaD), TCA cycle enzymes (citrate synthase), enzymes linked to fatty acid (FadB) or amino acid (aspartate aminotransferase) catabolism, and two putative membrane proteins that were, based upon their sequences, unlike previously characterized proteins. Given the magnitude of their mutant's defect, the aspartate aminotransferase, RNA helicase, and one of the putative membrane proteins were the factors most critical for L. pneumophila low-temperature growth. Thus, L. pneumophila not only employs some of the same processes and factors as other bacteria do in order to survive at low temperatures (e.g., LpxP, CsdA) but it also appears to possess novel modes of cold adaptation.
Legionella pneumophila is a gram-negative γ-proteobacterium that is ubiquitous in natural and man-made water systems [17, 29, 45, 60]. In its aquatic habitats, it exists planktonically, as an intracellular parasite of protozoa, and as a component of biofilms [35, 39, 45]. However, the ubiquity of L. pneumophila is also due to its capacity to survive at many temperatures, including those between 63°C and 4°C [17, 29, 56, 60]. Beyond its natural niche, L. pneumophila is also a human pathogen, whereby the inhalation of contaminated water droplets from aerosol-generating devices results in Legionnaires' disease . Given that L. pneumophila is transmitted to humans directly from water sources, it is important to understand how the legionellae survive in water, including how they adapt to high and low temperature conditions in order to survive in both natural habitats throughout the year, from the warmer to colder months, and man-made systems that provide both warm and cold water. Since L. pneumophila grows best in the laboratory at 32 to 37°C, there are many studies devoted to understanding its physiology at the higher temperatures . In contrast, studies on L. pneumophila at low temperatures are sparse, even though a large number of studies have identified factors that facilitate the low-temperature growth of other bacteria [7, 9, 43]. Like observations made for others, legionellae growing at low-temperatures have a greater amount of unsaturated lipid in their membranes and a need for the cytoplasmic ribonuclease R (RNase R) [8, 41]. However, it has also been documented that type II protein secretion (T2S) is critical for the survival and growth of L. pneumophila at 12-25°C, whether it be in rich broth, on agar media, in tap water, or in amoebae [53, 55, 56]. The T2S system of L. pneumophila secretes more than 25 proteins, including a wide variety of degradative enzymes and virulence factors as well as proteins that are more highly expressed at low temperatures [12, 14, 48, 54]. Finally, a secreted peptidyl-prolyl isomerase (PpiB), whose secretion is not dependent upon T2S or the known type IV secretion systems of L. pneumophila, is required for optimal growth at 17°C . In this study, we used a genetic screen to identify new genes that are specifically required for L. pneumophila growth at low-temperature.
L. pneumophila strain 130b (ATTC strain BAA-74) served as our wild-type strain . A lspF mutant of 130b (NU275) defective for T2S was previously described . Legionellae were cultured on buffered charcoal yeast extract (BCYE) agar at 37°C . Bacteria were also grown on casein and egg yolk plates to assess proteases and lipases [49, 55].
Strain 130b was randomly mutagenized with mini-Tn10, as previously described . Mutant colonies were then replica-plated onto two sets of BCYE agar. One set was incubated at 37°C for 3 days, whereas the other was incubated at 17°C for 6 days. Mutants that were impaired for growth specifically at the lower temperature compared to parental wild type were retested. To that end, bacteria taken from a 3-day old, 37°C BCYE plates were suspended in water, diluted, and plated for isolated colonies on BCYE agar incubated at 37°C and 17°C. The efficiency of plating was determined by dividing the number of CFU obtained at 17°C at days 20-21 by the number of CFU obtained at 37°C on day-3 .
To determine the site of the mini-Tn10 insertion in the various mutants, inverse PCR was performed  and then the reaction products were subjected to sequence analysis at our biotechnology facility. DNA and protein sequences obtained were used to search the L. pneumophila gene database (http:genolist.pasteur.fr/LegioList/). Annotations given in the L. pneumophila database were confirmed by searching the NCBI database.
To identify L. pneumophila genes that are important for low-temperature growth, a population of randomly mutagenized legionellae was screened for mutants that showed a reduced ability to grow at 17°C on BCYE agar. The temperature 17°C was chosen for several reasons. First, L. pneumophila is quite adept at surviving at 17°C in potable water . Second, growth kinetic, gene expression, and protein secretion patterns of L. pneumophila cultured 17°C are clearly distinguished from those occurring at the standard laboratory temperature of 37°C [54, 56]. Third, L. pneumophila is distinguished from some of the other Legionella species by its capacity to grow at 17°C . Fourth, as noted above, 17°C was an effective temperature for discerning the importance of L. pneumophila T2S and PpiB for both low-temperature growth and survival in water [54-56]. Finally, many of the observations made with wild type and T2S mutant L. pneumophila at 17°C could be extrapolated to other low temperatures, including 25°C, 17°C, 12°C, and 4°C [54-56]. From the 7400 colonies examined, eleven (mutants NU356 - NU366) exhibited a > 10-fold reduction (P < 0.05; Student's t-test) in efficiency of plating at 17°C compared to wild-type 130b (Table 1). All mutants grew comparably to wild type when incubated at 37°C on BCYE agar (data not shown). Based upon the magnitude their low-temperature growth defects, the mutants were divided into three groups (Table 1). The first three (NU356 - NU358) showed a relatively modest defect ranging roughly between 10- and 150-fold. The next six (NU359 - NU364) were severely impaired, exhibiting a 30,000- to 750,000-fold reduction in plating, an efficiency of plating that was comparable to that of previously defined T2S mutant NU275 (Table 1). The final two mutants (NU365, NU366) failed to form any colonies at 17°C. Inverse PCR and sequence analysis were done to identity of the genes that had been mutated.
Mutants NU361 and NU362 proved to be T2S mutants, containing insertions in lspE and lspGH (Table 1) and showing the expected lack of secreted proteolytic and lipolytic activities. Their severe growth defects were comparable to that of the lspF T2S mutant NU275 (Table 1). In many but not all gram-negative bacteria , T2S is a multi-step process in which proteins destined for export are translocated across the inner membrane in a Sec- or Tat-dependent manner, recognized in the periplasm, and then delivered to the T2S apparatus, whereupon a pilus-like structure “pushes” proteins through a dedicated outer membrane pore [12, 33]. The low-temperature growth defect of L. pneumophila T2S mutants is due, at least in part, to the loss of secreted proteins [54-56]. The importance of an intact T2S system for low-temperature growth has also been indicated for Shewanella oneidensis and Vibrio cholerae [22, 51]. That these two new T2S mutants were isolated validated our genetic screen.
Two of the new mutants (NU360, NU363) had their transposon insertion in a gene involved in the synthesis of lipid A (Table 1). NU360 and NU363, which were as defective as the T2S mutants, had their mutation in the same gene (lpg0363), albeit it in different locations. That two lpg0363 mutants were identified in the screen as well as the fact that lpg0363 is the last gene in an operon confirms the importance of this gene in the low-temperature growth of L. pneumophila. The lpg0363 gene encodes one of the three LpxL orthologs that occur in L. pneumophila . In E. coli, LpxL is an acyltransferase that attaches laurate to the lipid A precursor Kdo2-lipid IVA . However, E. coli also expresses an LpxL-related enzyme, namely LpxP, that attaches palmitoleate rather than laurate to the developing lipid A molecule. Importantly, in E. coli, whereas LpxL is important for lipid A synthesis at 30°C and above, LpxP is required for the production of cold-adapted lipid A and involved in cold-adapted replication [52, 58]. Recently, LpxP was found to be cold-induced in environmental S. oneidensis . Therefore, we hypothesize that lpg0363 encodes the L. pneumophila equivalent of LpxP (Table 1). Finding that a gene involved in lipid synthesis is required for L. pneumophila growth at lower temperatures is entirely compatible with what is known about cold adaptation in bacteria. Indeed, one of the most commonly reported cold adaptations is a change in lipid composition that serves to make bacterial membranes more fluid at lower temperatures [7, 9, 40, 43, 50]. This form of acclimation is often marked by an increase in the levels of unsaturated fatty acids, such as was discerned from previous biochemical work on L. pneumophila strain Corby grown at 24°C vs. 37°C . In the current situation, increases in unsaturation occur when palmitoleate, an unsaturated fatty acyl chain, replaces laurate in the lipid A moiety [40, 58]. Other cold-associated, lipid changes that have been defined in bacteria include a shortening of the fatty acid chains and the incorporation of branched and cyclic fatty acids [9, 43]. However, compared to most gram-negatives, Legionella species have a relatively high level of branched fatty acids, even when grown at 37°C [34, 59]. In sum, our data implicate an LpxP-like protein and an increase in unsaturated lipid A as important aspects of L. pneumophila growth at 17°C.
Two of the mutants (NU359, NU365) identified in our screen had insertions in genes involved in RNA degradation (Table 1). NU359, which was as impaired as the T2S and lpg0363 mutants were at 17°C, had its mutation in rnr (lpg0092), the gene encoding RNase R  (Table 1). The importance of RNAse R for the growth of L. pneumophila at low-temperatures was also documented when rnr mutants of the Philadelphia-1 strain exhibited impaired growth at 25 and 30°C . A link between RNAse R and growth at low-temperature has also been seen for other bacteria, including Aeromonas, Bacillus, Escherichia, and Pseudomonas [5, 6, 16, 19, 47]. RNAse R degrades RNA with extensive structure in the absence of helicase function and ATP [9, 10, 47]. Unlike NU359, NU365 was completely unable to grow at 17°C and had its insertion in lpg2345, the csdA (deaD) gene encoding an RNA helicase  (Table 1). A link between the CsdA/DeaD helicase and low-temperature growth was previously established when E. coli csdA mutants were found to have a reduced ability to grow at 15°C . CsdA/DeaD conjoins with ribonuclease E to form a “cold shock degradosome” . That csdA is monocistronic in L. pneumophila argues for the growth defect of NU365 being solely due to the lack of the helicase. We posit that CsdA/DeaD and RNAse R promote cold adaptation in L. pneumophila in the same way that they act in E. coli. When bacteria are exposed to cold, there is a stabilization of secondary structures in RNA and this in turn creates a transient inhibition of protein synthesis, but helicases and nucleases act to overcome this inhibition and thereby adapt to low temperature.
Three of the mutants (NU357, NU358, NU366) that had difficulty growing at 17°C had mutations in genes involved in central intermediary metabolism (Table 1). NU357 had an insertion in lpg1415, the gltA gene encoding citrate synthase, which catalyzes the first step in the TCA cycle, namely the formation of citrate from oxaloacetate and acetyl-CoA . Since gltA is monocistronic, the defect in NU357 is likely due to loss of citrate synthase. A link between gltA and cold adaptation is also suggested by the fact that the gene is cold-inducible in other bacteria . NU366 had its insertion in lpg1459, encoding aspartate aminotransferase which converts aspartate and α-ketoglutarate to oxaloacetate and glutamate . Loss of oxaloacetate and, to a lesser degree, glutamate would reduce the activity of the TCA cycle . In L. pneumophila, there are four ways to generate oxaloacetate, with the three other sources originating from malate, pyruvate, and p-enol pyruvate . Since NU366 was completely unable to grow at 17°C (Table 1), but appeared normal at 37°C, Lpg1459 seems to be a major means for generating oxaloacetate at low-temperature and a minor source at elevated temperatures. NU358 had its mutation in lpg1352 (fadB), whose product catalyzes the second and third steps in degradation (i.e., β-oxidation cycle) of imported medium- and long-chain fatty acids . The gene downstream of fadB is fadA, which encodes the enzyme responsible for the final step in β-oxidation of fatty acids . Thus, the defect of NU358 is most likely due to loss of fadB and fadA. In L. pneumophila, acetyl-CoA produced by the FadB- and FadA-mediated degradation of fatty acids feeds directly into the TCA cycle [13, 18, 27]. Although the TCA cycle has been viewed as a main means by which L. pneumophila assimilates carbon and produces energy when growing at 37°C [27, 32], the fact that three mutants lacking TCA cycle function were identified implies that the TCA cycle is even more important at low temperatures. That NU358 and NU366 were more impaired at 17°C than was the gltA mutant (Table 1) suggested that FadBA, aspartate aminotransferase, and/or their reaction products are involved in more processes that are important for low-temperature growth. In L. pneumophila, P. putida, and others, acetyl-CoA, whether a product of fatty acid or pyruvate (made from oxaloacetate) oxidation, can be used to make poly-3-hydroxybutyrate (PHB) [2, 24, 37, 38, 41]. Although PHB is best known as a carbon and energy reserve for bacteria growing in low-nutrient environments [21, 28, 31], it has also been shown that legionellae contain increased levels of PHB when grown at 24°C vs. 30°C and 37°C . PHB and related compounds have been recently implicated in the low-temperature adaptation of other bacteria, including Pseudomonas sp. 14-3 and Colwellia psychrerythraea [4, 42]. Thus, we posit that PHB may be another factor that is important for the ability of L. pneumophila to grow at 17°C.
NU364, which had a very severe growth defect at 17°C, had an insertion in lpg1755, annotated as encoding a 143.7-kDa transmembrane protein of unknown function (Table 1). Proteins showing similarity to Lpg1755 are predicted in the genomes of many other environmental bacteria (Table 2). The lpg1755 ORF is 10 bp downstream of an operon (fliFGHIJ) encoding flagellar proteins . Although it is unknown if Lpg1755 has any connection to flagella, the expression of flagella by L. pneumophila and others, such as Listeria monocytogenes, does increase as temperature drops from 37°C to 25-30°C [26, 36]. NU356, which had a more modest defect, had its insertion in a monocistronic gene (lpg1159) predicted to encode a 38.2-kDa metabolite transporter / membrane permease (Table 1). Hypothetical proteins similar to Lpg1159 (e.g., E values approaching 5 × 10-55) were also identified by BLASTP as being encoded by many environmental bacteria (data not shown). Membrane proteins, transporters, and permeases are among the factors induced at low temperatures in various bacteria, including Bacillus subtilis, S. oneidensis, and V. cholerae, [3, 5, 20]. These data suggest that the Lpg1755 and Lpg1159 proteins may represent two new but conserved aspects of bacterial low-temperature growth.
From the data obtained in this study as well as the results from past studies, a picture of how L. pneumophila adapts to low temperatures is beginning to emerge. Some of the processes implicated in low-temperature growth in other bacteria have now been documented in L. pneumophila, including increased levels of unsaturated fatty acids and lipid A and degradation of RNA. As for the factors involved in these processes, we can cite the LpxP ortholog, RNAse R, and the CsdA RNA helicase. Based on the size of the growth defect of the null mutants, the RNA helicase is particularly important. On the other hand, our study of L. pneumophila has uncovered newer aspects of low-temperature growth, including T2S, secreted peptidyl-prolyl isomerase, the TCA cycle, fatty acid and amino acid catabolism, as well as previously unidentified (membrane) proteins. Among these, the putative aminotransferase and membrane protein encoded by lpg1459 and lpg1755 appear to be especially critical, given the very severe, low-temperature growth defects of their mutants. Although our genetic screen utilized 17°C, we strongly suspect that, based upon our past work on T2S [53-56], the genes identified in the current study will prove to also be significant for L. pneumophila growth or survival, both extra- and intracellular, at other low temperatures. Further characterization of L. pneumophila mutants and proteins should increase our understanding of bacterial physiology at low-temperature as well as potentially offer insight into transmission of an important pathogen.
This work was supported by NIH grant AI43987 awarded to N. P. C.