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.

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.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors.

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis.

Paracoccus contaminans sp. nov., isolated from a contaminated water microcosm.

A beige-pigmented, oxidase-positive bacterial strain (WPAn02T), isolated as a presumably airborne contaminant of an axenic bacterial microcosm, was studied using a polyphasic taxonomic approach. Cells of the isolate were coccoid and stained Gram-negative. A comparison of the 16S rRNA gene sequence of strain WPAn02T with sequences of type strains of the most closely related species of the genus Paracoccus showed highest sequence similarities to Paracoccus chinensis (97.7 %), Paracoccus marinus (97.1 %), Paracoccus niistensis (97.4 %) and 'Paracoccus zhejiangensis' (97.0 %). 16S rRNA gene sequence similarities to all other species of the genus Paracoccuswere below 97 %. The fatty acid profile of the strain consisted of the major fatty acids C18 : 1ω7c/ω9t/ω12t and C18 : 0. DNA-DNA hybridizations between WPAn02T and type strains of P. chinensis, P. marinus, P. niistensis, and 'P. zhejiangensis' resulted in similarity values of 49 % (reciprocal 22 %), 16 % (reciprocal 10 %), 30 % (reciprocal 32 %), and 18 % (reciprocal 7 %), respectively. DNA-DNA hybridization results together with the differentiating biochemical and chemotaxonomic properties indicated that WPAn02T represents a novel species of the genus Paracoccus, for which the name Paracoccus contaminans sp. nov. (type strain WPAn02T=RKI 16-01929T=LMG 29738T=CCM 8701T=CIP 111112T), is proposed.

Genome Sequence of Paracoccus contaminans LMG 29738T, Isolated from a Water Microcosm.

We announce here the complete genome sequence of Paracoccus contaminans LMG 29738T, which we recently isolated from a contaminated water microcosm. The genome consists of a 2.94-Mb chromosome and a 94-kb plasmid. To our knowledge, we provide the first DNA methylation analysis of a Paracoccus species.