Jesus M. Eraso

The Early Divisome Protein FtsA Interacts Directly through Its 1c Subdomain with the Cytoplasmic Domain of the Late Divisome Protein FtsN

In Escherichia coli, FtsN localizes late to the cell division machinery, only after a number of additional essential proteins are recruited to the early FtsZ-FtsA-ZipA complex. FtsN has a short, positively charged cytoplasmic domain (FtsN(Cyto)), a single transmembrane domain (FtsN(TM)), and a periplasmic domain that is essential for FtsN function. Here we show that FtsA and FtsN interact directly in vitro. FtsN(Cyto) is sufficient to bind to FtsA, but only when it is tethered to FtsN(TM) or to a leucine zipper. Mutation of a conserved patch of positive charges in FtsN(Cyto) to negative charges abolishes the interaction with FtsA. We also show that subdomain 1c of FtsA is sufficient to mediate this interaction with FtsN. Finally, although FtsN(Cyto-TM) is not essential for FtsN function, its overproduction causes a modest dominant-negative effect on cell division. These results suggest that basic residues within a dimerized FtsN(Cyto) protein interact directly with residues in subdomain 1c of FtsA. Since FtsA binds directly to FtsZ and FtsN interacts with enzymes involved in septum synthesis and splitting, this interaction between early and late divisome proteins may be one of several feedback controls for Z ring constriction.

Interacting regulatory networks in the facultative photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1.

Regulation of photosynthetic membrane synthesis in Rhodobacter sphaeroides 2.4.1 is dependent on the interactions of numerous regulatory elements, with two of the most important being the cbb(3) terminal oxidase and the PrrBAC two-component regulatory system. Here, we reveal that the cbb(3) terminal oxidase possesses extensive, additional regulatory activities under anaerobic conditions, and that the PrrBAC system is further involved in the regulation of the expression of more than 20% of the R. sphaeroides genome under anaerobic conditions, extending well beyond functions related to redox gene expression.

The highly conserved MraZ protein is a transcriptional regulator in Escherichia coli

The mraZ and mraW genes are highly conserved in bacteria, both in sequence and in their position at the head of the division and cell wall (dcw) gene cluster. Located directly upstream of the mraZ gene, the Pmra promoter drives the transcription of mraZ and mraW, as well as many essential cell division and cell wall genes, but no regulator of Pmra has been found to date. Although MraZ has structural similarity to the AbrB transition state regulator and the MazE antitoxin and MraW is known to methylate the 16S rRNA, mraZ and mraW null mutants have no detectable phenotypes. Here we show that overproduction of Escherichia coli MraZ inhibited cell division and was lethal in rich medium at high induction levels and in minimal medium at low induction levels. Co-overproduction of MraW suppressed MraZ toxicity, and loss of MraW enhanced MraZ toxicity, suggesting that MraZ and MraW have antagonistic functions. MraZ-green fluorescent protein localized to the nucleoid, suggesting that it binds DNA. Consistent with this idea, purified MraZ directly bound a region of DNA containing three direct repeats between Pmra and the mraZ gene. Excess MraZ reduced the expression of an mraZ-lacZ reporter, suggesting that MraZ acts as a repressor of Pmra, whereas a DNA-binding mutant form of MraZ failed to repress expression. Transcriptome sequencing (RNA-seq) analysis suggested that MraZ also regulates the expression of genes outside the dcw cluster. In support of this, purified MraZ could directly bind to a putative operator site upstream of mioC, one of the repressed genes identified by RNA-seq.

Transcriptome response to nitrosative stress in Rhodobacter sphaeroides 2.4.1.

The facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1 has a nitric oxide-response transcriptional regulator, NnrR, and nitric oxide reductase (NOR), although it is incapable of denitrification. To investigate at the genomic level the physiological response to nitrosative stress of R. sphaeroides, the transcriptome profiles of strain 2.4.1 and its NnrR mutant were analyzed before and after exposure to nitrosating agents, S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), under microaerobic conditions. GSNO and SNP affected the expression of different but overlapping sets of genes. Only a limited number of these genes, including the genes for NOR, were under the control of NnrR, and those genes were significantly upregulated by GSNO and by SNP. The oxygen-responsive regulator FnrL and a predicted iron-sensing regulator were perhaps also involved in the transcriptome response to reactive nitrogen species. Some genes, including hemN for heme biosynthesis, were subject to dual regulation by NnrR and FnrL.

Redox flow as an instrument of gene regulation.

Publisher Summary Rhodobacter sphaeroides is a gram-negative facultative photoheterotroph with a branched electron transport chain (ETC), similar to the ETC of Paracoccus denitrificans . In the presence of oxygen, R. sphaeroides uses an ETC similar to the mitochondrial ETC as well as that of other proteobacteria. A number of genes involved in the redox control of gene expression in R. sphaeroides may possess homologs in higher eukaryotes, which suggests the existence of common evolutionary pathways able to affect the regulation of gene expression. This chapter describes methods and approaches used to study those elements involved in redox sensing in R. sphaeroides and describes how redox flow can serve as an instrument of gene expression. It investigates the many genetic approaches applicable to the study of R. sphaeroides , together with the extensive physiologic and biophysical studies of this organism, to explain the redox control of gene expression.

Transcriptional regulation of photosynthesis operons in Rhodobacter sphaeroides 2.4.1.

Publisher Summary Rhodobacter sphaeroides 2.4.1 is a gram-negative bacterium that, in addition to its ability to carry out anoxygenic photosynthesis (PS), has the capacity to grow by both aerobic and anaerobic respiration, fermentatively, and lithotrophically. The photosystem of R. sphaeroides 2.4.1 consists of three pigmentprotein complexes, comprising the reaction center (RC), and two light-harvesting (LH) complexes, designated B800-850 (LHII) and B875 (LHI), based on their respective absorption maxima. This chapter focuses on the methodological approaches that have employed to study the transcriptional regulation of those operons encoding the structural polypeptides comprising these complexes, as well as those required for the biosynthesis of the photopigments, carotenoids (Crts), bacteriochlorophyll (Bchl), and bacteriopheophytin. The chapter describes various approaches that have been successfully utilized to identify and analyze factors involved in regulating transcription of the PS operons. It provides a summary of those regulatory genes that have been identified and thus far characterized using these approaches.

Bacterial Cell Wall: Thinking Globally, Actin Locally

The bacterial actin-like protein MreB is thought to form a continuous helical polymer at the membrane to confer rod shape. Two new studies now show that MreB forms discrete dynamic patches that travel circumferentially.

Genomic landscape of intrahost variation in Group A Streptococcus: repeated and abundant mutational inactivation of the fabT gene encoding a regulator of fatty acid synthesis.

ABSTRACT To obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here.

Gene fitness landscape of group A streptococcus during necrotizing myositis

Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified novel GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon directed insertion-site sequencing (TraDIS) we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins, and exporters for toxins, quorum sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding five transporters confirmed that each isogenic mutant strain was significantly impaired in causing necrotizing myositis in NHPs. qRT-PCR analysis showed that these five genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface-exposed, suggesting that our findings have translational research implications.