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Changes in relative transcript amounts caused by ∆pbp1a, double ∆pbp1a mltG(∆488bp), and triple ∆pbp1a mltG(∆488bp) ∆pbp2b mutations in Streptococcus pneumoniae

ABSTRACT: In ellipsoid-shaped ovococcus bacteria, such as the respiratory pathogen Streptococcus pneumoniae (pneumococcus), side-wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin-binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG-domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ΔmltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. To gain insight into the mechanism of synthetic-viable genetic relationships involving Δpbp1a, we performed RNA-Seq analyses of Δpbp1a, Δpbp1a mltG(Δ488bp), and Δpbp1a mltG(Δ488bp) Δpbp2b mutants compared to the isogenic unencapsulated parent strain. Unexpectedly, the Δpbp1a deletion causes an almost exclusive induction in relative transcript amounts of genes that are in the WalRK TCS regulon, which includes established and putative PG hydrolases and division proteins of unknown functions.

ORGANISM(S): Streptococcus pneumoniae

PROVIDER: GSE77021 | GEO | 2016/04/19

SECONDARY ACCESSION(S): PRJNA309290

REPOSITORIES: GEO

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Project description:Streptococcus pneumoniae is a human commensal bacterium that causes serious diseases. Peptidoglycan (PG) is the critical component of bacteria that maintains cell shape, size, chaining, and turgor resistance. Because of its surface exposure and eubacterial-specific mechanism of biosynthesis, PG biosynthesis remains an outstanding target for discovery of new antibiotics to resistant “superbugs,” like S. pneumoniae. Pneumococcal PG biosynthesis is carried out by a balance of septal and peripheral (side-wall-like) PG synthesis mediated by bPBP2x and bPBP2b, respectively, that emanates from midcell regions. We selected for additional suppressor mutations, besides mltG, that eliminate the requirement for essential bPBP2b in peripheral PG synthesis. We found that mutations that inactivate a putative RNA binding protein, designated KhpA, suppressed the requirement for some, but not all of the essential proteins required for peripheral PG synthesis. KhpA was used as bait in co-IP experiments to demonstrate interactions with a second RNA binding protein, designated KhpB, in cells. Mutations in khpA or khpB phenocopy each other and result in slower growth rates, smaller cell size with normal aspect ratios, and induction of cell wall stress responses. RIP-Seq analyses confirmed KhpAB as RNA binding protein in cells. Another suppressor of Δpbp2b implicated induction of a cell division operon as a way to bypass the requirement for bPBP2b. We show that increased expression of this cell division operon occurs in ΔkhpAB mutants and that this induction is necessary and sufficient to account for suppression of Δpbp2b. Comparisons of relative transcript amounts and protein levels indicate that induction of this cell division operon in ΔkhpAB mutants occurs primarily at the post-transcriptional level. Together, our results show that KhpAB, which are virulence factors in S. pneumoniae and are conserved in other Gram-positive pathogens, acts as a new class of RNA binding protein, with properties analogous to Hfq in Gram-negative bacteria.

Project description:Background: Responses to hypoxia have been investigated in many species; however, comparative study between conspecific geographical populations in different altitude regions is rare, especially for invertebrates . The migratory locust, Locusta migratoria, is widely distributed both on high-altitude Tibetan Plateau (TP) and on low-altitude North China Plain (NP). TP locusts have inhabited Tibetan Plateau since Quaternary glaciations events and thus probably have evolved superior capacity to deal with hypoxia. Results: Here we compared the hypoxic responses of TP and NP locusts from morphological, behavioral and physiological perspectives. We found that TP locusts were more tolerant of extreme hypoxia than NP locusts, with a lower proportion exhibiting stupor, a faster recovery time, and higher respiration rates. We compared the transcriptional profiles of field TP and NP locusts and found that their differences were possibly attributed to a combination of multiple factors, e.g. oxygen, UV radiation, temperature and nutrition. To evaluate why TP locusts respond to extreme hypoxia differently from NP locusts, we subjected them to extreme hypoxia and compared their transcriptional responses. We found that the aerobic metabolism was more active in TP locusts than in NP locusts. RNAi disruption of PDHE1b, an entry gene from glycolysis to TCA cycle, increased the ratio of stupor in Tibetan locusts and decreased the ATP content of Tibetan locusts in hypoxia, confirming the significant importance of this metabolic branch for TP locusts to conquer hypoxia. Conclusions: Here we show that TP locusts are better tolerant of hypoxia than NP locusts and the better capacity to modulate primary metabolism in TP locusts contributes to their superior tolerance of hypoxia compared to NP locusts. FIELD POPULATION: TP locusts vs. NP locusts;direct comparison on 6 separate microarrays; each microarray compares one biological replicate; each biological replicate contains 10 individuals. LAB POPULATION: hypoxia-treated TP locusts vs TP locusts in normoxia; hypoxia-treated NP locusts vs NP locusts in normoxia; direct comparison on 6 separate microarrays; each microarray compares one biological replicate; each biological replicate contains 10 individuals.

Dataset Information

Changes in relative transcript amounts caused by ∆pbp1a, double ∆pbp1a mltG(∆488bp), and triple ∆pbp1a mltG(∆488bp) ∆pbp2b mutations in Streptococcus pneumoniae

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