A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis (Mtb) is caused by mutations in restricted regions of the genome. Mutations in katG, the promoter region of the mabA–inhA operon, and inhA are those most frequently responsible for isoniazid (INH) resistance. Several INH‐resistant (INH^r) Mtb clinical isolates without mutations in these regions have been described, however, indicating that there are as yet undetermined mechanisms of INH resistance. We identified the mabA^g609a silent mutation in a significant number of INH^r Mtb clinical isolates without known INH resistance mutations. A laboratory strain, H37Rv, constructed with mabA^g609a, was resistant to INH. We show here that the mabA^g609a mutation resulted in the upregulation of inhA, a gene encoding a target for INH, converting the region adjacent to the mutation into an alternative promoter for inhA. The mabA^g609a silent mutation results in a novel mechanism of INH resistance, filling in a missing piece of INH resistance in Mtb.     

Biotreatment of restaurant wastewater with an oily high concentration by newly isolated bacteria from oily sludge

DNA methylation has been introduced as a promising biomarker for different diseases. Alterations in macrophage DNA methylation status have been documented during Mycobacterium tuberculosis (Mtb) infection. We conducted this study using a human methylation PCR array kit, which comprised a panel of 22 genes in TLR2 signaling pathway, in order to gain insights into epigenetic interactions between drug-susceptible and -resistant Mtb strains and THP-1-derived macrophages (one of the main host immunity cells during TB infection). We also evaluated the expression of Rv1988 gene in the studied isolates. It was found that the methylation level of all of the studied inflammatory genes, except Irak-2 and Tbk-1, increased in THP-1 macrophages, which were infected by extensively drug-resistant (XDR) Mtb strains, compared with the mock cells (P?<?0.05). In susceptible strains, we only found hypomethylation in Irak-2 gene, in addition to a slight increase in the methylation levels of Ubev, Ube2n, and Traf6 genes. The present findings provide new insights into the potential role of resistant and susceptible Mtb strains in promoting aberrant epigenetic modifications in macrophages. Further investigations on the host epigenomes, infected with different Mtb isolates, are needed to elucidate their functions in immunological responses and to introduce new effective tools against Mtb infection.

     

Selective autophagy gets more selective: Uncoupling of autophagy flux and xenophagy flux in Mycobacterium tuberculosis-infected macrophages

Induction of autophagy has been reported as a potential means to eliminate intracellular pathogens. Corroborating that, many studies report inhibition of autophagy as a survival strategy of bacterial pathogens. Incidentally, autophagy at the basal level is critical for survival of host cells including macrophages. We asked how a bacterial pathogen could inhibit autophagy for its survival if the inhibition resulted in cell death. In a recent study we show distinct regulation of autophagy in Mycobacterium tuberculosis (Mtb)-infected macrophages where Mtb containing- and nonMtb-containing autophagosomes show different fates in terms of maturation. We show that upon Mtb infection, there is no dramatic change in the autophagy flux in macrophages. However, autophagosomes that contain the virulent strains of Mtb show selective resilience to the maturation phase of autophagy. Surprisingly, nonMtb-containing autophagosomes in the infected cells continue to mature into autolysosomes. The block in the xenophagy flux is missing in the case of avirulant infections. We show that this selectivity is achieved through selective exclusion of RAB7 from virulent Mtb-containing autophagosomes, thereby restricting the formation of amphisomes.     

Functional characterization of hypothetical proteins of Mycobacterium tuberculosis with possible esterase/lipase signature: a cumulative in silico and in vitro approach

The functional aspect of several mycobacterium proteins annotated as hypothetical are yet to be discovered. In the present investigation, in silico approaches were used to predict the biological function of some of the unknown Mtb proteins, which were further validated by wet lab experiments. After screening thousands of Mtb proteins, functionally unknown hypothetical proteins Rv0421c, Rv0519c, Rv0774c, Rv1191, Rv1592c, and Rv3591c were chosen on the basis of their importance in Mtb life cycle. All these proteins posses the α/β-hydrolase topological fold, characteristic of lipases/esterases, with serine, aspartate, and histidine as the putative members of the catalytic triad. The catalytic serine is located in pentapeptide motif “GXSXG” and oxyanion residue is in dipeptide motif HG. To further support our observation, molecular docking was performed with conventional synthetic lipolytic substrates (pNP-esterss) and specific lipase/esterase inhibitors (tetrahydrolipstatin and phenylmethanesulfonyl fluoride (PMSF)). Significant docking score and strong interaction of substrates/inhibitors with these proteins revealed that these could be possible lipases/esterases. To validate the in silico studies, these genes were cloned from Mtb genome and the proteins were over-expressed in pQE-30/Escherichia coli M15 system. The expressed proteins were purified to homogeneity and enzymatic activity was determined using pNP esters as substrate. The enzyme activity of recombinant proteins was inhibited by tetrahydrolipstatin and PMSF pre-treatment. Outcome of the present investigation provided a basic platform to analyze and characterize unknown hypothetical proteins.     

Depletion of the DarG antitoxin in Mycobacterium tuberculosis triggers the DNA-damage response and leads to cell death

Of the ~80 putative toxin-antitoxin (TA) modules encoded by the bacterial pathogen Mycobacterium tuberculosis (Mtb), three contain antitoxins essential for bacterial viability. One of these, Rv0060 (DNA ADP-ribosyl glycohydrolase, DarG_Mtb), functions along with its cognate toxin Rv0059 (DNA ADP-ribosyl transferase, DarT_Mtb), to mediate reversible DNA ADP-ribosylation (Jankevicius et al., 2016). We demonstrate that DarT_Mtb-DarG_Mtb form a functional TA pair and essentiality of darG_Mtb is dependent on the presence of darT_Mtb, but simultaneous deletion of both darT_Mtb-darG_Mtb does not alter viability of Mtb in vitro or in mice. The antitoxin, DarG_Mtb, forms a cytosolic complex with DNA-repair proteins that assembles independently of either DarT_Mtb or interaction with DNA. Depletion of DarG_Mtb alone is bactericidal, a phenotype that is rescued by expression of an orthologous antitoxin, DarG_Taq, from Thermus aquaticus. Partial depletion of DarG_Mtb triggers a DNA-damage response and sensitizes Mtb to drugs targeting DNA metabolism and respiration. Induction of the DNA-damage response is essential for Mtb to survive partial DarG_Mtb-depletion and leads to a hypermutable phenotype.     

Impact of bedaquiline and capreomycin on the gene expression patterns of multidrug-resistant Mycobacterium tuberculosis H37Rv strain and understanding the molecular mechanism of antibiotic resistance

The emergence of multidrug resistance (MDR), extensively drug-resistant, and total drug-resistant Mycobacterium tuberculosis (Mtb) strains have hampered the treatment of tuberculosis (TB). Capreomycin and Bedaquiline are currently used for MDR-TB treatment. To understand the impact of these antibiotics on Mtb genes, we have curated the gene expression data where the Mtb cultures were exposed to the Bedaquiline and Capreomycin. Based on the P value cut off (<0.05) and logFC (<−0.5 and >+0.5) values, we have selected the top differentially expressed genes during the antibiotic exposures. We have observed that the top differentially expressed Mtb genes were related to universal stress genes, two-component regulatory systems, and drug efflux pumps. We have curated the Mtb gene datasets and carried out the functional over-representation analysis using the individual gene expression values. We further, constructed the gene interaction networks of antibiotic resistance genes and virulence genes of Mtb to understand the impact of the antibiotics at the molecular level and thus to understand the antimicrobial resistance and virulence patterns. Our study elucidates the impact of antibiotics on the Mtb genes at the molecular level and the positively enriched pathways, operons, and regulons data are helpful in understanding the resistance patterns in Mtb. The upregulated genes during the exposure of Bedaquiline and Capreomycin can be considered as potent drug targets for the development of new anti-TB drugs.     

Toxicity of chlorpyrifos‐methyl to Sitophilus zeamais collected in Korea and biochemical differences

In this study, lethal concentration (LC₅₀) values of chlorpyrifos‐methyl (CPM) were determined for two Korean strains (CBNU and KNU) of Sitophilus zeamais. The two strains had similar susceptibilities (1.70 and 1.86 μg a.i./cm², respectively) to CPM. Carboxylesterase (CE) activity was twice as high in the CBNU strain as in the KNU strain. Lower acetylcholinesterase (AChE) activity was also noted in the latter; however, the activity of glutathione S‐transferase (GST) was twice as high as in the CBNU strain. Gel electrophoresis of CE of crude extracts from adults of the two strains of S. zeamais showed clearly different band patterns, with molecular weights of 60 kDa and 71 kDa in the CBNU and KNU strains, respectively. MALDI‐TOF MS/MS was used to profile small proteins (less than 10 kDa), with results indicating that 206 proteins are expressed differently in the two strains. The peak of interest of 2247.7 m/z was applied to TOF‐TOF MS and its de novo peptide sequence was identified as a tyrosine phosphatase fragment. Phospholipids from the two strains were analyzed and 34 phospholipids were found to be significantly different between strains. Results suggest that the two strains collected from Korea showed different biochemical results, presumably differences in insecticide selection by different living locations.     

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome–lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C‐terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non‐pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.     

利用基因组学和MALDI-TOF MS技术鉴定放线菌纲细菌的核糖体蛋白质标志物

【目的】基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法基于微生物的特征蛋白指纹图谱鉴定菌种,本研究利用基因组学和MALDI-TOFMS技术鉴定放线菌纲细菌的核糖体蛋白质标志物。【方法】从MALDI-TOF MS图谱数据库选取放线菌纲代表菌种,在基因组数据库检索目标菌种,获取目标菌株或其参比菌株的核糖体蛋白质序列,计算获得分子质量理论值,用于注释目标菌株MALDI-TOFMS指纹图谱中的核糖体蛋白质信号。【结果】从8目,24科,53属,114种,142株放线菌的MALDI-TOFMS图谱中总共注释出31种核糖体蛋白质。各菌株的指纹图谱中核糖体蛋白质信号数量差异显著。各种核糖体蛋白质信号的注释次数差异显著。总共15种核糖体蛋白质在超过半数图谱中得到注释,注释次数最高的是核糖体大亚基蛋白质L36。【结论】本研究找到了放线菌纲细菌MALDI-TOF MS图谱中常见的15种核糖体蛋白质信号,可为通过识别核糖体蛋白质的质谱特征峰鉴定放线菌的方法建立提供依据。     

Genotypic and phenotypic characteristics of tunisian isoniazid-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Forty three isoniazid (INH)-resistant Mycobacterium tuberculosis isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA −15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and sequencing reaction. Mutations at either codon were detected in 67.5% of isolates: katG315 in 37.2, mabA −15C→T in 27.9 and both of them in 2.4%, respectively. katG sequencing showed a G insertion at codon 325 detected in 2 strains and leading to amino acid change T326D which has not been previously reported. Distribution of each mutation, among the investigated strains, showed that katG S315T was associated with multiple-drug profile, high-level INH resistance and loss or decreased catalase activity; whereas the mabA −15C→T was more prevalent in mono-INH resistant isolates, but it was not only associated with a low-level INH resistance. It seems that determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most clinical INH-resistant strains.     

Comparative proteome analysis of porcine follicular fluid and serum reveals that excessive α2‐macroglobulin in serum hampers successful expansion of cumulus‐oocyte complexes

Porcine follicular fluid (pFF) constitutes the micro‐environment of the maturing oocyte. Although pFF is a transudate of serum, in pigs, it is superior to serum in promoting in vitro expansion of the cumulus cells, a specialized cell population surrounding the oocyte. A comparative proteome analysis of autologous serum and pFF was performed to investigate proteins involved in successful cumulus expansion of porcine oocytes. iTRAQ labeling followed by 2‐D LC ESI‐Q‐TOF MS/MS revealed 63 proteins common to both fluids of which the abundance of 13 proteins was significantly different (p<0.05). Seven proteins were more concentrated in serum whereas six proteins were more abundant in pFF. To investigate the importance of these proteins, the cumulus matrices of COCs were collected after in vitro maturation in media supplemented with either of both biologically fluids and then subjected to 2‐D PAGE analysis. α₂‐Macroglobulin and CH4 and secrete domains of swine IgM, which were both less abundant in pFF, were absent from cumulus matrix extracts after in vitro maturation in pFF. Although both proteins were incorporated in the matrices of cumulus‐oocyte complexes matured in serum, depletion of α₂‐macroglobulin from serum could significantly compensate for the impaired cumulus expansion of oocytes matured in serum.     

Molecular analysis of the prokaryotic ubiquitin‐like protein (Pup) conjugation pathway in Mycobacterium tuberculosis

Proteins targeted for degradation by the Mycobacterium proteasome are post‐translationally tagged with prokaryotic ubiquitin‐like protein (Pup), an intrinsically disordered protein of 64 residues. In a process termed ‘pupylation’, Pup is synthesized with a terminal glutamine, which is deamidated to glutamate by Dop (deamidase of Pup) prior to attachment to substrate lysines by proteasome accessory factor A (PafA). Importantly, PafA was previously shown to be essential to cause lethal infections by Mycobacterium tuberculosis (Mtb) in mice. In this study we show that Dop, like PafA, is required for the full virulence of Mtb. Additionally, we show that Dop is not only involved in the deamidation of Pup, but also needed to maintain wild‐type steady state levels of pupylated proteins in Mtb. Finally, using structural models and site‐directed mutagenesis our data suggest that Dop and PafA are members of the glutamine synthetase fold family of proteins.     

IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.     

Multiple deletions in the polyketide synthase gene repertoire of Mycobacterium tuberculosis reveal functional overlap of cell envelope lipids in host–pathogen interactions

Several specific lipids of the cell envelope are implicated in the pathogenesis of M. tuberculosis (Mtb), including phthiocerol dimycocerosates (DIM) that have clearly been identified as virulence factors. Others, such as trehalose‐derived lipids, sulfolipids (SL), diacyltrehaloses (DAT) and polyacyltrehaloses (PAT), are believed to be essential for Mtb virulence, but the details of their role remain unclear. We therefore investigated the respective contribution of DIM, DAT/PAT and SL to tuberculosis by studying a collection of mutants, each with impaired production of one or several lipids. We confirmed that among those with a single lipid deficiency, only strains lacking DIM were affected in their replication in lungs and spleen of mice in comparison to the WT Mtb strain. We found also that the additional loss of DAT/PAT, and to a lesser extent of SL, increased the attenuated phenotype of the DIM‐less mutant. Importantly, the loss of DAT/PAT and SL in a DIM‐less background also affected Mtb growth in human monocyte‐derived macrophages (hMDMs). Fluorescence microscopy revealed that mutants lacking DIM or DAT/PAT were localized in an acid compartment and that bafilomycin A1, an inhibitor of phagosome acidification, rescued the growth defect of these mutants. These findings provide evidence for DIM being dominant virulence factors that mask the functions of lipids of other families, notably DAT/PAT and to a lesser extent of SL, which we showed for the first time to contribute to Mtb virulence.     

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 Å resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T‐loop of one subunit and the C‐loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T‐loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T‐loop makes direct contact with the γ‐phosphate of the ATP molecule replacing the Mg²⁺ position seen in the Methanococcus jannaschii GlnK1 structure. The C‐loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C‐terminal 3₁₀ helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.     

Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis

Isoniazid (INH) resistance of the Mycobacterium tuberculosis Complex (MtbC) is associated with both loss of catalase activity and mutation of the inhA gene. However, the relative contributions of these changes to resistance and to the loss of virulence for guinea-pigs is unknown. In this study, a virulent strain of Mycobacterium bovis, a member of the MtbC., was exposed to increasing concentrations of INH. Two INH-resistant strains were produced which had lost catalase activity. Strain WAg405, which had a higher resistance to INH, also had a mutation in the inhA gene. This demonstrated that loss of catalase activity and mutation of inhA had a cumulative effect on INH resistance. When a functional katG gene was integrated into the genome of WAg405 the INH resistance was greatly reduced. This indicated that most of the resistance had been caused by loss of catalase activity. While the parent INH-sensitive strain was virulent for guinea-pigs, the INH-resistant strains were significantly less virulent. Integration of a functional katG gene into the most resistant strain restored full virulence. This clearly established that katG is a virulence factor for M. bovis and that mutation of the inhA gene has no effect on virulence.     

Whole Cell Screen for Inhibitors of pH Homeostasis in Mycobacterium tuberculosis

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) encounter acidic microenvironments in the host and must maintain their acid-base homeostasis to survive. A genetic screen identified two Mtb strains that cannot control intrabacterial pH (pH_IB) in an acidic environment; infection with either strain led to severe attenuation in mice. To search for additional proteins that Mtb requires to survive at low pH, we introduced a whole-cell screen for compounds that disrupt pH_IB, along with counter-screens that identify ionophores and membrane perturbors. Application of these methods to a natural product library identified four compounds of interest, one of which may inhibit novel pathway(s). This approach yields compounds that may lead to the identification of pathways that allow Mtb to survive in acidic environments, a setting in which Mtb is resistant to most of the drugs currently used to treat tuberculosis.     

Mycobacterium tuberculosis RpfE crystal structure reveals a positively charged catalytic cleft

Resuscitation promoting factor (Rpf) proteins, which hydrolyze the sugar chains in cell‐wall peptidoglycan (PG), play key roles in prokaryotic cell elongation, division, and escape from dormancy to vegetative growth. Like other bacteria, Mycobacterium tuberculosis (Mtb) expresses multiple Rpfs, none of which is individually essential. This redundancy has left unclear the distinct functions of the different Rpfs. To explore the distinguishing characteristics of the five Mtb Rpfs, we determined the crystal structure of the RpfE catalytic domain. The protein adopts the characteristic Rpf fold, but the catalytic cleft is narrower compared to Mtb RpfB. Also in contrast to RpfB, in which the substrate‐binding surfaces are negatively charged, the corresponding RpfE catalytic pocket and predicted peptide‐binding sites are more positively charged at neutral pH. The complete reversal of the electrostatic potential of the substrate‐binding site suggests that the different Rpfs function optimally at different pHs or most efficiently hydrolyze different micro‐domains of PG. These studies provide insights into the molecular determinants of the evolution of functional specialization in Rpfs.     

An Extracellular Disulfide Bond Forming Protein (DsbF) from Mycobacterium tuberculosis: Structural, Biochemical, and Gene Expression Analysis

Disulfide bond forming (Dsb) proteins ensure correct folding and disulfide bond formation of secreted proteins. Previously, we showed that Mycobacterium tuberculosis DsbE (Mtb DsbE, Rv2878c) aids in vitro oxidative folding of proteins. Here, we present structural, biochemical, and gene expression analyses of another putative Mtb secreted disulfide bond isomerase protein homologous to Mtb DsbE, Mtb DsbF (Rv1677). The X-ray crystal structure of Mtb DsbF reveals a conserved thioredoxin fold although the active-site cysteines may be modeled in both oxidized and reduced forms, in contrast to the solely reduced form in Mtb DsbE. Furthermore, the shorter loop region in Mtb DsbF results in a more solvent-exposed active site. Biochemical analyses show that, similar to Mtb DsbE, Mtb DsbF can oxidatively refold reduced, unfolded hirudin and has a comparable pK_a for the active-site solvent-exposed cysteine. However, contrary to Mtb DsbE, the Mtb DsbF redox potential is more oxidizing and its reduced state is more stable. From computational genomics analysis of the M. tuberculosis genome, we identified a potential Mtb DsbF interaction partner, Rv1676, a predicted peroxiredoxin. Complex formation is supported by protein coexpression studies and inferred by gene expression profiles, whereby Mtb DsbF and Rv1676 are upregulated under similar environments. Additionally, comparison of Mtb DsbF and Mtb DsbE gene expression data indicates anticorrelated gene expression patterns, suggesting that these two proteins and their functionally linked partners constitute analogous pathways that may function under different conditions.     

Growth,osmotic downshock resistance and differentiation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains lacking mechanosensitive channels

Previous work has shown that the mechanosensitive (MS) channel of large conductance (MscL) is essential for preventing lysis of Bacillus subtilis log phase cells upon a rapid, severe osmotic downshock. Growing cells of B. subtilis strains lacking MscL and one or more putative MS channel proteins of small conductance (YhdY, YkuT and YfkC) showed even higher sensitivity to an osmotic downshock. The effect was greatest for a strain lacking MscL and YkuT, and a strain lacking all four MS channel proteins had a similar phenotype. These defects were complemented by expression of either MscL or YkuT in trans. All MS channel mutant strains ultimately became resistant to osmotic downshock in stationary phase but at varying times, with mscL ykuT strains taking the longest time to become resistant. Expression of β-galactosidase from gene fusions to lacZ showed modest expression of ykuT and lower levels of expression of yhdY and yfkC when strains were grown in medium containing high salt. Sporulation of all MS channel mutant strains was normal, and the mutant spores germinated normally with l-alanine or dodecylamine.