glnA Truncation in Salmonella enterica Results in a Small Colony Variant Phenotype, Attenuated Host Cell Entry, and Reduced Expression of Flagellin and SPI-1-Associated Effector Genes

Abstract

Many pathogenic bacteria use sophisticated survival strategies to overcome harsh environmental conditions. One strategy is the formation of slow-growing subpopulations termed small colony variants (SCVs). Here we characterize an SCV that spontaneously emerged from an axenic Salmonella enterica serovar Typhimurium water culture. We found that the SCV harbored a frameshift mutation in the glutamine synthetase gene glnA, leading to an ∼90% truncation of the corresponding protein. Glutamine synthetase, a central enzyme in nitrogen assimilation, converts glutamate and ammonia to glutamine. Glutamine is an important nitrogen donor that is required for the synthesis of cellular compounds. The internal glutamine pool serves as an indicator of nitrogen availability in Salmonella In our study, the SCV and a constructed glnA knockout mutant showed reduced growth rates, compared to the wild type. Moreover, the SCV and the glnA mutant displayed attenuated entry into host cells and severely reduced levels of exoproteins, including flagellin and several Salmonella pathogenicity island 1 (SPI-1)-dependent secreted virulence factors. We found that these proteins were also depleted in cell lysates, indicating their diminished synthesis. Accordingly, the SCV and the glnA mutant had severely decreased expression of flagellin genes, several SPI-1 effector genes, and a class 2 motility gene (flgB). However, the expression of a class 1 motility gene (flhD) was not affected. Supplementation with glutamine or genetic reversion of the glnA truncation restored growth, cell entry, gene expression, and protein abundance. In summary, our data show that glnA is essential for the growth of S. enterica and controls important motility- and virulence-related traits in response to glutamine availability.IMPORTANCESalmonella enterica serovar Typhimurium is a significant pathogen causing foodborne infections. Here we describe an S Typhimurium small colony variant (SCV) that spontaneously emerged from a long-term starvation experiment in water. It is important to study SCVs because (i) SCVs may arise spontaneously upon exposure to stresses, including environmental and host defense stresses, (ii) SCVs are slow growing and difficult to eradicate, and (iii) only a few descriptions of S. enterica SCVs are available. We clarify the genetic basis of the SCV described here as a frameshift mutation in the glutamine synthetase gene glnA, leading to glutamine auxotrophy. In Salmonella, internal glutamine limitation serves as a sign of external nitrogen deficiency and is thought to regulate cell growth. In addition to exhibiting impaired growth, the SCV showed reduced host cell entry and reduced expression of SPI-1 virulence and flagellin genes.

Keywords: SPI-1; Salmonella enterica; glutamine synthetase; motility; small colony variant.

FIG 1

Attenuated growth of the S. Typhimurium SCV and a glnA knockout mutant, compared to the wild type and a revertant. (A) Colony morphology on LB agar. (B) Growth curves for growth in LB broth. (C) Growth behavior on M9G minimal medium. Data shown in panel B are means from quadruplicate cultures for which OD₆₀₀ values were read at 15-min intervals, and all data are representative of three independent experiments. Wt, wild type; SCV, small colony variant; Rev, revertant; ΔglnA, glnA knockout mutant.

FIG 2

Reading frame truncation in the glutamine synthetase gene glnA, triggering the S. Typhimurium SCV phenotype. The addition of glutamine restores SCV and glnA mutant growth. (A) Genetic characteristics of the wild-type, SCV, and revertant strains with regard to the glutamine synthetase gene glnA. The numbering of nucleotides is relative to the translational start site. Insertion of adenosine 120 in the glnA gene of S. Typhimurium SCV (boxed in black) causes a frameshift mutation, resulting in a premature stop codon (marked by an asterisk). The revertant shows a compensatory deletion of cytosine 123 (boxed in gray), leading to restoration of the glnA open reading frame. (B) Growth behavior of the S. Typhimurium strains in liquid LB medium supplemented with 2 mM l-glutamine (+ Gln). Growth of the SCV and ΔglnA mutant was restored to wild-type and revertant levels, respectively, when glutamine was added to the LB broth. The results were derived from the same experiment as shown in Fig. 1B, with the difference that l-glutamine was added to the growth medium. Data shown are means from quadruplicate cultures for which OD₆₀₀ values were read at 15-min intervals, and data are representative of three independent experiments. (C) Growth of the SCV and ΔglnA mutant, compared to the wild type and revertant, respectively, on 2 mM l-glutamine-supplemented M9G minimal medium (+ Gln). Wt, wild type; SCV, small colony variant; Rev, revertant; ΔglnA, glnA knockout mutant.

FIG 3

Felix O1 phage resistance phenotype of S. Typhimurium SCV and a glnA knockout mutant, compared to the wild type and a revertant. (Upper) In the absence of glutamine (− Gln), the wild type and revertant were susceptible to Felix O1 phage, whereas the SCV and a glnA knockout mutant were resistant. (Lower) In the presence of 2 mM l-glutamine (+ Gln), all strains were susceptible to Felix O1 phage. Data are representative of at least three independent experiments. Wt, wild type; SCV, small colony variant; Rev, revertant; ΔglnA, glnA knockout mutant.

FIG 4

Reduced entry of the S. Typhimurium SCV and a glnA knockout mutant in U937 cells. Entry is restored in the revertant strain or when strains are grown in culture medium with glutamine before infection. CFU were determined after a 30-min entry period and 90 min of gentamicin killing of extracellular bacteria. (A) Entry of the wild type, SCV, and revertant grown without glutamine and the SCV grown with 2 mM l-glutamine (+G) in U937 cells. (B) Entry of the S. Typhimurium wild type, a glnA knockout mutant grown without glutamine, and a glnA knockout mutant grown in the presence of 2 mM l-glutamine (+G) in U937 cells. Data are representative of at least three independent experiments. *, significantly reduced number of intracellular bacteria, compared to the wild type (P < 0.02, Student's two-tailed t test, type 2). Wt, wild type; SCV, small colony variant; Rev, revertant; ΔglnA, glnA knockout mutant.

FIG 5

Severely reduced levels of flagellin and SPI-1-associated effectors in the SCV and a glnA knockout mutant. Protein levels are restored when strains are grown in culture medium with glutamine. (A and B) Coomassie blue-stained SDS-PAGE gels of early-stationary-phase bacteria grown in LB broth. (A) Extracellular (left) and whole-cell (right) proteins of wild-type, SCV, and glnA knockout mutant strains grown without or with (+G) l-glutamine supplementation. (B) S. Typhimurium ATCC 14028s wild-type and isogenic glnA, invC (nonfunctional SPI-1-dependent secretion system), and ssaV (nonfunctional SPI-2-dependent secretion system) knockout mutant strains grown without glutamine and a glnA knockout mutant strain grown with the addition of 2 mM l-glutamine (+G). (C and D) Western blot analysis of the extracellular and whole-cell protein fractions, as described above, with flagellin-specific antibodies (C) and SopA-, SopB-, and SopD-specific antibodies (D). Negative controls were the ΔfliGHI strain for antiflagellin blots, strain M712 for anti-SopA/anti-SopB blots, and strain MvP1895 for anti-SopD blots. Lanes on the left show prestained protein ladders; the values at the left indicate kilodaltons. Wt, wild type; SCV, small colony variant; ΔglnA, glnA knockout mutant; 14028s, ATCC 14028s; control, negative control.

FIG 6

Reduced expression of flagellin genes, the class 2 motility gene flgB, and several SPI-1-associated effector genes in the SCV and the glnA mutant, compared to the wild type and a genetic revertant. Analysis of transcript levels in different S. Typhimurium strain backgrounds was performed by semiquantitative RT-PCR, using gene-specific primers for the flagellin-encoding genes fliC and fljB (class 3 motility genes) (A), the SPI-1 effector genes sopA, sopB, and sopD (B), and the class 2 motility gene flgB and the class 1 motility gene flhD (C). Transcript levels of S. Typhimurium gyrB, the constitutively expressed gene encoding gyrase subunit B, were used as the control. Images represent agarose gels of ethidium bromide-stained PCR products after different rounds of amplification. Genes whose transcript levels differed markedly in the different strain backgrounds are marked in gray. Data are representative of three independent experiments. Wt, wild type; SCV, small colony variant; Rev, revertant; glnA, glnA knockout mutant.

FIG 7

Model of the flagellar gene network and SPI-1-associated effector expression in the S. Typhimurium SCV and a glnA mutant. Functional impairment of glnA in the SCV or a glnA knockout mutant results in glutamine limitation and negatively affects flagellum biosynthesis and SPI-1-mediated virulence. Specifically, reduced expression of class 2 (flgB) and class 3 (fliC and fljB) motility genes and SPI-1-associated effector genes was detected, accompanied by reduced abundance of flagellin and effector proteins (depicted by orange arrows). The dashed orange lines outline the different possible ways of influencing flagellar and SPI-1-associated effector expression upstream of the class 2 motility genes. It is established that the flagellar and SPI-1 regulons are interconnected by the class 2 motility gene product FliZ, which activates SPI-1 via the HilD regulatory protein (45, 51). Thus, reduced levels of FliZ are expected to lead to reduced levels of the transcriptional regulators HilA (controlled by HilD) and InvF (controlled by HilA), both of which are known to activate the expression of effector genes located inside and outside (indicated by a dotted box) SPI-1 (77–79). While transcriptional regulation of sopA and sopB by HilA/InvF has been described in the literature (78, 80, 81), sopD regulation is less well understood (indicated by a dashed black arrow). HBB, hook basal body; SCV, small colony variant; SPI-1, Salmonella pathogenicity island 1; T3SS-1, SPI-1-encoded type III secretion system; σ⁷⁰, housekeeping sigma factor; σ²⁸, flagellum-specific alternative sigma factor (FliA).