Legionella pneumophila usurps host cell lipids for vacuole expansion and bacterial growth.

Vacuolar pathogens reside in membrane-bound compartments within host cells. Maintaining the integrity of this compartment is paramount to bacterial survival and replication as it protects against certain host surveillance mechanisms that function to eradicate invading pathogens. Preserving this compartment during bacterial replication requires expansion of the vacuole membrane to accommodate the increasing number of bacteria, and yet, how this is accomplished remains largely unknown. Here, we show that the vacuolar pathogen Legionella pneumophila exploits multiple sources of host cell fatty acids, including inducing host cell fatty acid scavenging pathways, in order to promote expansion of the replication vacuole and bacteria growth. Conversely, when exogenous lipids are limited, the decrease in host lipid availability restricts expansion of the replication vacuole membrane, resulting in a higher density of bacteria within the vacuole. Modifying the architecture of the vacuole prioritizes bacterial growth by allowing the greatest number of bacteria to remain protected by the vacuole membrane despite limited resources for its expansion. However, this trade-off is not without risk, as it can lead to vacuole destabilization, which is detrimental to the pathogen. However, when host lipid resources become extremely scarce, for example by inhibiting host lipid scavenging, de novo biosynthetic pathways, and/or diverting host fatty acids to storage compartments, bacterial replication becomes severely impaired, indicating that host cell fatty acid availability also directly regulates L. pneumophila growth. Collectively, these data demonstrate dual roles for host cell fatty acids in replication vacuole expansion and bacterial proliferation, revealing the central functions for these molecules and their metabolic pathways in L. pneumophila pathogenesis.

Functional analysis of ars gene cluster of Pannonibacter indicus strain HT23(T) (DSM 23407(T)) and identification of a proline residue essential for arsenate reductase activity.

Arsenic is a naturally occurring ubiquitous highly toxic metalloid. In this study, we have identified ars gene cluster in Pannonibacter indicus strain HT23(T) (DSM 23407(T)), responsible for reduction of toxic pentavalent arsenate. The ars gene cluster is comprised of four non-overlapping open reading frames (ORFs) encoding a transcriptional regulator (ArsR), a low molecular weight protein tyrosine phosphatases (LMW-PTPase) with hypothetical function, an arsenite efflux pump (Acr3), and an arsenate reductase (ArsC). Heterologous expression of arsenic inducible ars gene cluster conferred arsenic resistance to Escherichia coli ∆ars mutant strain AW3110. The recombinant ArsC was purified and assayed. Site-directed mutagenesis was employed to ascertain the role of specific amino acids in ArsC catalysis. Pro94X (X = Ala, Arg, Cys, and His) amino acid substitutions led to enzyme inactivation. Circular dichroism spectra analysis suggested Pro94 as an essential amino acid for enzyme catalytic activity as it is indispensable for optimum protein folding in P. indicus Grx-coupled ArsC.

Pannonibacter indica sp. nov., a highly arsenate-tolerant bacterium isolated from a hot spring in India.

A novel aerobic bacterium, strain HT23(T), able to grow on 500 mM sodium arsenate was isolated from a hot-spring sediment sample collected from Athamallik, Orissa, India. Cells of this isolate were Gram negative. Heterotrophic growth was observed at pH 6.0-11.0 and 20-45 °C. Optimum growth was observed at 37 °C and pH 7.0-10.0. The major polar lipids are diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl monomethyl ethanolamine. The major isoprenoid quinone was Q-10. 16S rRNA gene sequence analysis indicated that the bacterium clustered with the genus Pannonibacter and showed 98.9 % similarity with Pannonibacter phragmitetus C6-19(T) (DSM 14782(T)) and 98 % with the P. phragmitetus group B and P. phragmitetus group E strains. Levels of DNA-DNA relatedness between the strain HT23(T) and P. phragmitetus C6-19(T) (DSM 14782(T)) and other strains of P. phragmitetus group B and group E strains were below 55 %. On the basis of phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA-DNA hybridization data, strain HT23(T) is considered to represent a novel species of the genus Pannonibacter, for which the name Pannonibacter indica sp. nov. is proposed. The type strain is HT23(T) (=JCM 16851(T) = DSM 23407(T) = LMG 25769(T)).

Microbacterium oryzae sp. nov., an actinobacterium isolated from rice field soil.

A novel aerobic soil actinobacterium (strain MB10(T)) belonging to the genus Microbacterium was isolated from rice field soil samples collected from Jagatpur, Orissa, India. Cells were Gram-stain positive, short rod-shaped and motile. The strain was oxidase-negative and catalase-positive. Heterotrophic growth was observed at pH 5.0-11.0 and at 16-37 °C; optimum growth was observed at 28 °C and pH 7.0-9.0. The DNA G+C content was 71.6 mol%. Predominant cellular fatty acids of strain MB10(T) were iso-C14 : 0, anteiso-C15 : 0, C16 : 0, iso-C16 : 0 and anteiso-C17 : 0. Cell wall sugars were galactose, glucose and rhamnose. The major isoprenoid quinones were MK-9 (10 %), MK-10 (43 %) and MK-11 (36 %). The peptidoglycan represents the peptidoglycan type B2ß. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phospholipid and unknown glycolipids. 16S rRNA gene sequence identity revealed the strain MB10(T) clustered within the radiation of the genus Microbacterium and showed 99.2 % similarity with Microbacterium barkeri DSM 20145(T). However, DNA-DNA similarity study was 37.0 % with Microbacterium barkeri DSM 20145(T), the nearest phylogenetic relative. On the basis of phenotypic and chemotaxonomic properties, 16S rRNA gene sequence analysis and DNA-DNA reassociation studies, it is proposed that strain MB10(T) represents a novel species of the genus Microbacterium, for which the name Microbacterium oryzae sp. nov. is proposed; the type strain is MB10(T) ( = JCM 16837(T) = DSM 23396(T)).

Mutation in the lysA gene impairs the symbiotic properties of Mesorhizobium ciceri.

A Tn5-induced mutant of Mesorhizobium ciceri, TL28, requiring the amino acid lysine for growth on minimal medium was isolated and characterized. The Tn5 insertion in the mutant strain TL28 was located on a 6.8-kb EcoRI fragment of the chromosomal DNA. Complementation analysis with cloned DNA indicated that 1.269 kb of DNA of the 6.8-kb EcoRI fragment restored the wild-type phenotype of the lysine-requiring mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to lysA gene of different bacteria. The lys (-) mutant TL28 was unable to elicit development of effective nodules on the roots of Cicer arietinum L. There was no detectable level of lysine in the root exudates of chickpea. However, addition of lysine to the plant growth medium restored the ability of the mutant to produce effective nodules with nitrogen fixation ability on the roots of C. arietinum.

Thiosulfate oxidation by Comamonas sp. S23 isolated from a sulfur spring.

A bacterial isolate S23 capable of oxidizing thiosulfate was isolated from a sulfur spring. Strain S23 is gram-negative, aerobic, and motile. The G + C content of DNA is 61.4 mol%. The fatty acid composition and phylogenetic analysis of the 16S rRNA gene sequence of strain S23 showed that it is related to the members of the genus Comamonas, and most closely related to Comamonas testosteroni (99.9% sequence similarity). The isolate S23 exhibited thiosulfate oxidation under a mixotrophic growth condition. Polymerase chain reaction (PCR) using soxB-specific primers and DNA sequencing showed the presence of the soxB gene. This is the first report in Comamonas sp. showing thiosulfate oxidation under a mixotrophic growth condition.

VIPER regulates naive T cell activation and effector responses: Implication in TLR4 associated acute stage T cell responses.

Naive T cells are known to express the modest level of TLR4 while it is known to go down during TCR activation. However, information towards the requirement of TLR4 signaling during TCR or mitogenic activation of naive wild-type T cells remains scanty. Here we have investigated the endogenous functional expression of TLR4 in naive mice T cells during TCR and mitogenic stimulation in presence of VIPER peptide (VP), an established inhibitor of TLR4 signaling. As expected we found that TLR4 expression goes down during TCR and mitogenic activation. Interestingly, we observed that VP treatment restores TLR4 expression on those activated T cells. Moreover, VP was found to regulate such activation of naive T cell as evident by reduction of CD25, CD69 expression, effector cytokines (IL-2, IFN-γ, TNF) production, T cell proliferation and down-regulation of T cell activation-dependent Fas (CD95), FasL (CD95L) expression. Together, our current observation highlights a possible requirement of TLR4 responses in T cells, which might have possible implication towards the pathogenic acute phase activation of naive T cells.

Draft Genome Sequence of Pannonibacter indicus Strain HT23T (DSM 23407T), a Highly Arsenate-Tolerant Bacterium Isolated from a Hot Spring in India.

Pannonibacter indicus strain HT23T, a highly arsenate-tolerant bacterium, was isolated from a tropical hot spring. The estimated genome is 4.2 Mb with 3,818 protein-coding sequences containing putative genes, some of which are involved in arsenate resistance.