An Expanded Genetic Toolbox to Accelerate the Creation of Acholeplasma laidlawii Driven by Synthetic Genomes.

We have developed genetic tools for the atypical bacterium Acholeplasma laidlawii. A. laidlawii is a member of the class Mollicutes, which lacks cell walls, has small genomes, and has limited metabolic capabilities, requiring many metabolites from their hosts. Several of these traits have facilitated the development of genome transplantation for some Mollicutes, consequently enabling the generation of synthetic cells. Here, we propose the development of genome transplantation for A. laidlawii. We first investigated a donor-recipient relationship between two strains, PG-8A and PG-8195, through whole-genome sequencing. We then created multihost shuttle plasmids and used them to optimize an electroporation protocol. We also evolved a superior strain for DNA uptake via electroporation. We created a PG-8A donor strain with a Tn5 transposon carrying a tetracycline resistance gene. These tools will enhance Acholeplasma research and accelerate the effort toward creating A. laidlawii strains with synthetic genomes.

The Functionality of IbpA from Acholeplasma laidlawii Is Governed by Dynamic Rearrangement of Its Globular-Fibrillar Quaternary Structure.

Small heat shock proteins (sHSPs) represent a first line of stress defense in many bacteria. The primary function of these molecular chaperones involves preventing irreversible protein denaturation and aggregation. In Escherichia coli, fibrillar EcIbpA binds unfolded proteins and keeps them in a folding-competent state. Further, its structural homologue EcIbpB induces the transition of EcIbpA to globules, thereby facilitating the substrate transfer to the HSP70-HSP100 system for refolding. The phytopathogenic Acholeplasma laidlawii possesses only a single sHSP, AlIbpA. Here, we demonstrate non-trivial features of the function and regulation of the chaperone-like activity of AlIbpA according to its interaction with other components of the mycoplasma multi-chaperone network. Our results show that the efficiency of the A. laidlawii multi-chaperone system is driven with the ability of AlIbpA to form both globular and fibrillar structures, thus combining functions of both IbpA and IbpB when transferring the substrate proteins to the HSP70-HSP100 system. In contrast to EcIbpA and EcIbpB, AlIbpA appears as an sHSP, in which the competition between the N- and C-terminal domains regulates the shift of the protein quaternary structure between a fibrillar and globular form, thus representing a molecular mechanism of its functional regulation. While the C-terminus of AlIbpA is responsible for fibrils formation and substrate capture, the N-terminus seems to have a similar function to EcIbpB through facilitating further substrate protein disaggregation using HSP70. Moreover, our results indicate that prior to the final disaggregation process, AlIbpA can directly transfer the substrate to HSP100, thereby representing an alternative mechanism in the HSP interaction network.

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii.

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with KD ~ 1 µM. Proteins co-localize in the soluble fraction of the cell at 30-37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpAHis6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30-37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.

Cell-in-Cell Phenomena in Wall-Less Bacteria: Is It Possible?

This work describes curious structures formed by the mainly phytopathogenic mycoplasma Acholeplasma laidlawii, as well as the human pathogen Ureaplasma parvum cells which resemble cell-in-cell structures of higher eukaryotes and protists. The probable significance of such structures for the mycoplasma cell is discussed. The possibility of their formation in nature and their potential role in the transformation of genetic material, for example, by maintaining (on the one hand) the stability of the genome in the line of generations during asexual reproduction or (on the other hand) the genome plasticity, are substantiated. It should be especially noted that all the arguments presented are based only on morphological data. However, closer attention to unusual structures, the existence of which was shown by electron microscopy images in this case, may prompt researchers to analyze their data more carefully and find something rare and non-trivial among seemingly trivial things. If it is proven by additional methods that cell-in-cell structures can indeed be formed by prokaryotes without a cell wall, this phenomenon may acquire general biological significance.

Adaptation of Mycoplasmas to Antimicrobial Peptides: Development of Melittin Resistance in Acholeplasma laidlawii Is Associated with Changes in Genomic and Proteomic Profiles and in Virulence.

For the first time it is shown that the development of resistance to melittin in Acholeplasma laidlawii, a mycoplasma that is widely spread in nature and that is the main contaminant of cell cultures and vaccines, is associated with significant changes in the genomic profile, in cellular and vesicular proteomes, as well as in virulence.

[Experimental estimation of proteome size for cells and human plasma].

Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70,000 proteoforms, and plasma--1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)--40,000, E. coli--6200, P. furiosus--3400. In hepatocytes, the amount of proteoforms was the same as in HepG2--70,000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.

The Escherichia coli Envelope Stress Sensor CpxA Responds to Changes in Lipid Bilayer Properties.

The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.

[Effect of heat shock on cells of phytopathogenic mycoplasma Acholeplasma laidlawii PG-8A].

Heat shock caused a more active formation of the "dormant" forms (minibodies), as well as increased production of extracellular membrane vesicles by Acholeplasma laidlawii PG-8A cells. Raise of the amount of the minibodies that have increased resistance to biogenic and abiogenic stress factors and pathogenicity may lead to more successful persistence of mycoplasmas in their hosts. Increased production of the extracellular membrane vesicles containing virulence factors by Acholeplasma laidlawii cells during stress may be an additional burden for the infected organism. It has been recently revealed that the vesicles of A. laidlawii contain appreciable quantities of small heat shock protein IbpA (Hsp20). In this paper, using immune-electron microscopy, have shown that at elevated temperature IbpA is associated with A. laidlawii minibodies. Perhaps, IbpA contributes to increased resistance and pathogenicity of the minibodies, keeping their proteins and polypeptides, including protein virulence factors in the folding-competent state.

Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins.

Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen-host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects.

Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes.

The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.

The identification and characterization of IbpA, a novel α-crystallin-type heat shock protein from mycoplasma.

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.

The acylation state of surface lipoproteins of mollicute Acholeplasma laidlawii.

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.

[Comparative proteomic characteristic of Mycoplasmas (Mollycutes)].

Saturating proteome identification and the study of post-translational protein modifications of Acholeplasma laidlawii using combination of single- and two-dimension gel electrophoresis followed by mass-spectrometry analysis have been carried out. Results were compared to the earlier identified proteome of Mycoplasma gallisepticum. It was found that M. gallisepticum and A. laidlawii express 61 and 58% of the annotated ORFs respectively. All subunits of DNA-polymerase III were identified during our study which indicates that our methods can detect single copies of a given protein per cell. Metabolic pathways of the respective mycoplasmas were compared further in this work.

Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein.

HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.

[Oligomeric forms, functions and cellular localization of alpha-crystallin type protein from Acholeplasma laidlawii].

Alpha-Crystallin type heat shock protein (alpha-HSP) IbpA from Acholeplasma laidlawii was expressed in Escherichia coil and isolated from cell extract on Ni-sepharose column. Recombinant IbpA, like other alpha-HSPs, spontaneously formed oligomeres in vitro. High resolution electron microscopy revealed regular structures with 15 nm in diameter. Evaluation of molecular mass of IbpA oligomers was performed by gel filtration. Most of oligomers consist of 24 subunits. Recombinant IbpA prevents heat denaturation of soluble proteins in cell extract of E. coli and displays a mild positive effect on thermotolerance of E. coli cells during severe heat shock. We investigated a localization of IbpA in A. laidlawii cell by immunocytochemistry. We suppose that IbpA may protect various intracellular structures from damage during heat shock.

[The heat shock protein of alpha-crystallin type from mycoplasma (Acholeplasma laidlawii)].

A considerable increase in several heat shock proteins (HSPs) amount in Acholeplasma laidlawii cells has been revealed after temperature rising of liquid culture; and the quantity of small HSP, named p17, was increased in a hundred of times. The p17 protein was isolated and identified as HSP of alpha-crystallin type (alpha-HSP). It became possible as a result of sequencing of 15 amino acids from N-terminal of the p17 polypeptide chain, followed by revealing of a corresponding open reading frame (ORF) in a completely sequenced genome of A. laidlawii PG 8A. Computer-based search for homologous ORFs in all 17 genomes of Mycoplasmataceae family (the mycoplasmas themselves) that had been completely sequenced to date, gives negative result. But among the representatives of Mollicutes (mycoplasma) class, the genes coding alpha-HSPs were found in two Phytoplasma species (Phytoplasmataceae family) and the acholeplasma examined (Acholeplasmataceae family). It supposed that presence or absence of alpha-HSPs in microorganisms might be connected with the fact that representatives of Acholeplasmataceae and Phytoplasmataceae families inhabit principally in plant tissues in contrast to majority of Mycoplasmataceae family, that inhabit animal and human tissues, i. e. use ecological niches with relatively constant temperature.

[Effect of silver nitrate, adenosine-5'-monophosphate and phosphoenolpyruvate on activity of extracellular fructose bisphosphatase of Acholeplasma laidlawii var. granulum strain 118].

The reactions of glycolysis or gluconeogenesis proceed in good coordination in the cells of microorganisms, and each stage of these processes is distinctly regulated. Under such conditions fructose-bisphosphatase (FBPase) activity (the enzyme level being constant in the cells of microorganisms) is inhibited by adenosine-5'-monophosphate (AMP) and is activated by phosphoenolpyruvate (PEP) depending on the kind of the source of carbon (glycolytic or glyconeogenic) used for microorganism growth. It is evident that the corresponding regulation of FBPase should be absent in the extracellular environment where one cannot observe a distinct coordination of functioning of the enzyme systems. The investigation results prove that both AMP and PEP, under their individual testing in concentrations up to 20 microM did not practically affect activity of extracellular FBPase, and at higher concentrations they sharply decreased its activity (200 microM AMP by 70%, and PEP - by 75%). Under joint use of PEP and AMP (in concentration 200 microM and 500 microM) one could observe mutual neutralization of the effect of these substances on FBPase; as a result, its activity decreased only by 15% under AMP concentration of 500 microM, and by 25% at AMP concentration of 200 microM, that is in complete agreement with the data of individual testing of the above substances. PEP in high concentrations has displayed itself as a more active repressor of FBPase activity than AMP. AgNO3 in concentrations to 20 microM has manifested itself as a moderate stimulator of FBPase activity and even in the concentration of 200 microM it decreased the enzyme activity by 50% only. The data obtained are rather different than those described in literature for cellular FBPases of microorganisms. It is known that AMP is a powerful inhibitor of its FBPases activity (Ki = 5 microM) while PEP activates it (Ka = 20 microM).