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Appl Environ Microbiol. 2010 July; 76(14): 4905–4908.
Published online 2010 May 28. doi:  10.1128/AEM.01806-09
PMCID: PMC2901722

Acinetobacter baylyi Starvation-Induced Genes Identified through Incubation in Long-Term Stationary Phase[down-pointing small open triangle]

Abstract

Acinetobacter species encounter cycles of feast and famine in nature. We show that populations of Acinetobacter baylyi strain ADP1 remain dynamic for 6 weeks in batch culture. We created a library of lacZ reporters inserted into SalI sites in the genome and then isolated 30 genes with lacZ insertions whose expression was induced by starvation during long-term stationary phase compared with their expression during exponential growth. The genes encode metabolic, gene expression, DNA maintenance, envelope, and conserved hypothetical proteins.

Acinetobacter species are ubiquitous soil organisms. Starvation during long-term stationary phase (LTSP) can serve as a laboratory model for natural competitive conditions such as those found in soils (4). This model has been used to study Escherichia coli, and here, we have applied it to Acinetobacter baylyi strain ADP1 (8).

During long-term batch culture, an initially clonal population of Escherichia coli experiences five growth stages: lag, exponential, and stationary phases and then death phase and LTSP (4). Prior to LTSP, most of the cells die and serve as nutrition for starving survivors (6, 13). In LTSP, the cell population remains almost steady, declining slowly over years (reviewed in reference 4): for each newly dead cell, slightly less than one new cell is “born.”

Much of what is known about starvation physiology during LTSP has been determined through study of the growth advantage in stationary phase (GASP) phenotype. The phenotype arises from genetic changes that occur when cells experience LTSP. During LTSP, the population may have a mutation frequency approaching 1 in 600 base pairs per genome (5).

Some physiological changes that take place during LTSP have been described, as have some genes necessary for the development of GASP (13, reviewed in reference 12). Some mutant strains that exhibit GASP have mutations that enhance catabolic efficiency for processing amino acids (14-16). Another nutrient consumed is DNA, which requires genes homologous to strain ADP1's competence genes (6). Additionally, mutations that knock out SOS polymerases interfere with the formation of GASP mutants (11).

Growth of Acinetobacter baylyi.

One of our aims was to ascertain whether the phenomenon of LTSP occurs in A. baylyi. We cultured A. baylyi in aerated minimal succinate (0.01 M) broth (7) for 6 weeks at 37°C and measured the CFU ml−1 over that period (Fig. (Fig.1).1). Death phase began after about 24 h postinoculation, while LTSP began at 48 h postinoculation and persisted for at least five more weeks.

FIG. 1.
Growth of Acinetobacter baylyi ADP1 in minimal medium with 0.01 M succinate (7) over 6 weeks. Data are from a sample representative of three trials.

Identification of starvation-induced genes.

Our second aim was to identify ADP1 genes induced by starvation during LTSP. To do this, we followed the method of Chakravorty et al. (2), digesting ADP1 chromosomal DNA with SalI and ligating the DNA to a lacZ-kan cassette from plasmid pKOK6, resulting in circular DNA that could enter the ADP1 genome by a single-crossover event following natural transformation (see the supplemental material). We used a scaled-down version of the Miller method (9) in 96-well plates to screen 3,569 kanamycin-resistant colonies of ADP1 from six different ligation pools for mutants with higher levels of β-galactosidase activity during LTSP (48 h after inoculation) than during exponential phase (6 h after inoculation). There are 305 SalI sites in each genome (1), and most of the lacZ insertions were isolated from more than one independent pool, so the screen is near saturation.

One hundred twenty-three isolates were subjected to secondary screening and exhibited greater induction of β-galactosidase activity at 7 days postinoculation than during exponential phase. Examination of the strains using inverse PCR based on the sequence of the kanamycin cassette revealed 30 unique starvation-induced (STI) lacZ insertion sites (see the supplemental material).

The procedure used to introduce the lacZ-kan cassette into each strain should have resulted in merodiploid DNA caused by a single recombination event between the chromosome and a closed circle of DNA created by ligating SalI chromosomal fragments to SalI-digested cassette DNA. Using PCR, we detected an intact target open reading frame (ORF) in every strain. The insertion of the cassette may have resulted in chromosomal rearrangements, so we also investigated the chromosome structure surrounding the cassette. We used PCR to attempt to amplify the sequence between the DNA adjacent to the 5′ end of the lacZ-kan cassette and the lacZ gene and the sequence between the DNA adjacent to the 3′ end of the lacZ-kan cassette and the kan gene. Twenty-seven strains were merodiploids without any obvious nearby rearrangements. Three strains (AGCV5, AGCV7, and AGCV11) did not produce the expected PCR products, suggesting that they may have chromosomal rearrangements.

Expression of long-term stationary-phase genes.

All 30 STI genes were induced at 7 days postinoculation compared with their expression during exponential growth (Fig. (Fig.2B;2B; also see Table Table2).2). When we moved the kanamycin-linked lacZ reporters into a fresh ADP1 strain through natural transformation using boiled cell extracts as DNA donors and selection for kanamycin resistance, the reporter genes of all 30 new reporter strains were still induced in LTSP (data not shown).

FIG. 2.
(A) Growth of four representative lacZ reporter strains in minimal medium with 0.01 M succinate (7). Squares, ostA::lacZ strain; diamonds, pilU::lacZ strain; triangles, argS::lacZ strain; circles, tolB::lacZ strain. (B) Induction of β-galactosidase ...
TABLE 2.
Expression of STI gene-lacZ fusions after 7 days of incubation in batch culture

To further increase confidence in the identification of STI genes, we measured the population growth of null mutants (3) lacking one of six STI genes (ACIAD0167, ACIAD0615, ACIAD1960, ACIAD3229, or ACIAD3343, encoding conserved hypothetical proteins [CHPs], or pilU) or a control gene also encoding a CHP (ACIAD2370) but adjacent to and carried on the opposite strand relative to one of the STI genes. All of the strains grew normally in minimal broth Davis (BD, Franklin Lakes, NJ) supplemented with 0.01 M succinate for the first 48 h of incubation at 37°C with aeration (P > 0.1; unpaired, two-tailed Student's t test). All six strains except the ACIAD2370::kan strain, the negative control, showed a significant decrease in survival at 7 days (P < 0.05; data not shown).

Bioinformatic analysis of STI genes.

Twenty-eight of the STI lacZ reporters were inserted in an open reading frame (Table (Table1).1). Seven of these genes (23%) encode CHPs (3). Twenty other STI genes have annotations that rely on limited similarity, conserved amino acid motifs, or the function of homologous genes studied in a distant relative. The only STI gene in Acinetobacter with an experimentally determined phenotype is almA, which is required for the degradation of long-chain N-alkanes (10).

TABLE 1.
Bacterial strains isolated in this study

The expression of STI genes suggests that ADP1 cells in LTSP are metabolically active. We found specific translation-associated genes, such as fusA and infB, to be starvation induced (Tables (Tables11 and and2).2). Other STI genes encode anabolic proteins, such as those involved in amino acid synthesis (leuA and gshA), cell wall synthesis (kdsB), or catabolism (mdcA and almA). Three of the STI genes are annotated as encoding DNA maintenance, recombination, or competence proteins (Tables (Tables11 and and2),2), suggesting that, as in E. coli, control of DNA is important during LTSP (6, 11). A fourth group of A. baylyi STI genes encodes proteins that are associated with the bacterial envelope (Table (Table22).

Here, we have reported that strain ADP1 populations persist in LTSP. We have identified 30 genes that are induced in LTSP compared with their expression during exponential phase. The induced genes encode proteins needed for catabolism, anabolism (including protein synthesis), and control of DNA. Twenty-three percent of the induced genes encode CHPs, suggesting that these proteins should no longer be considered hypothetical. We conclude that ADP1 cells, like E. coli cells, are active during LTSP. Further scrutiny of ADP1's physiology during this model of feast-famine could help reveal how Acinetobacter species survive in highly competitive conditions such as those found in soils.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by a Howard Hughes Medical Institutes Undergraduate Science Education grant to Grinnell College, Patricia Armstrong Johnson Chair funds for B.A.V., and a Colorado College Natural Sciences Executive Committee grant to C.P.L.

We thank Leslie Gregg-Jolly, Grinnell College undergraduates Robin Lindemann, Ruth Emrick, Jessica Schmidt, Erin Schmidt, Paul Duffin, Felicia Barriga, and Lilliana Radoshevich, and Colorado College undergraduates Yuliya Muratov, Sarah Stanley, and Lucille Wenegieme.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 May 2010.

Supplemental material for this article may be found at http://aem.asm.org/.

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