Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation

Abstract

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the histone-like nucleoid-structuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis, as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Δrv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δrv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Δrv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis. We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein.IMPORTANCE Mycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not a typical nucleoid-associated protein (NAP) as expected from its initial annotation. Rv3852 from M. tuberculosis has neither influence on nucleoid shape or compaction nor a role in virulence. Our findings reduce the repertoire of identified nucleoid-associated proteins in M. tuberculosis to four transcription regulators and underline the importance of genetic studies to assign a function to bacterial genes. KEYWORDS tuberculosis, Mycobacterium tuberculosis, H-NS, NAP, global regulation, rv3852T he chromosome must be heavily compacted to fit into a bacterial cell about 1,000 times smaller than the length of its DNA. Similarly to eukaryotes, where DNA is wrapped around histones for condensation, prokaryotes possess nucleoid-associated proteins (NAPs), which influence DNA topology. Typically, NAPs interact with the chromosome at several hundred binding sites in a sequence-specific or non-sequencespecific manner (1). As a result, NAPs can bridge, loop, and bend DNA (2), thus

Polarly Localized EccE 1 Is Required for ESX-1 Function and Stabilization of ESX-1 Membrane Proteins in Mycobacterium tuberculosis

“…RpoB, an RNA polymerase subunit, was found mainly in the cytosol and partly in the membrane. Rv3852, a membrane protein, was present only in the membrane fraction, as previously described (30). EsxB, a substrate of the ESX-1 system, was detected in the cytosol and to a lesser extent in the capsule, although it was found mainly in the culture filtrate.…”

“…2999) were purchased from Cell Signaling. Polyclonal anti-Rv3852 antibody was produced by Eurogentec (30).…”

“…The M. tuberculosis ΔeccE 1 /eccE 1 HA mutant was grown in 100 ml of Sauton's medium without Tween 80 for 4 days with a starting OD 600 of 0.5. Bacteria were harvested by centrifugation, and the fractions were collected as described previously (30).…”

Essential Nucleoid Associated Protein mIHF (Rv1388) Controls Virulence and Housekeeping Genes in Mycobacterium tuberculosis

“…To probe where mIHF is localized inside the bacterial cell, M. tuberculosis H37Rv cell extracts were fractionated prior to subsequent immunoblotting. Proteins RpoB, Rv3852 17 and EsxB were used as positive controls for the cytosol, membrane and secreted fractions, respectively. The mIHF protein was detected in the cytosol only (Fig.…”

“…NAPs have a major effect on bacterial gene expression by acting as global transcription regulators and chromosome architects. In M. tuberculosis , three NAPs - EspR, Lsr2 and HupB - were characterized in depth previously, while the function of a fourth NAP candidate, Rv3852, was disproved 17 . In this report, the systematic investigation of mIHF is presented and its potential importance for M. tuberculosis pathogenesis described.…”